Wednesday, March 17, 2010

Types of OBJECTIVES for microscopes

OBJECTIVES:

The objectives are the most important component of an optical microscope because they determine the quality of images. There’re many types of objectives, achromatic, semi-plan, plan-achromatic and apochromatic are the most popular but some brands use their own description to identify best quality objectives like for Motic use EF Plan, etc…


The properties of each objective’s lens refer to aberrations or defects.
Chromatic aberration: color defect
Spherical aberration: shape defect on the image produced by the curve of lens.

Achromatic : the lens only correct the chromatic aberration, colors red and blue, but still give spherical aberration. It is the cheapest type lens most used in microscope construction.
Semi-plan : the lens corrects the chromatic aberration (red & blue) and spherical aberration. It is made of 2 lens, one concave and one convex.
E-Plan: deliver long working distances, high numerical aperture and flat images over the entire field of view with virtually no curvature of field. (E-Plan, Nikon nomenclature).
Plan-Achromatic : the lens corrects the chromatic aberration as well as provide excellent plan images without spherical aberration. It is usually made of 1 len without curve.
Plan-Apochromatic: is the best type of lens, corrects all chromatic aberrations: red, blue and green as well as spherical aberration, provides a plan image and wider visual field of view. It is used on lens of + 22mm field view or superior. (Zeiss has 25mm plan-apo)
Infinity Optical System: In modern and research microscopes the objective is designed to focuses an image to infinity. This permits to insert additional accessories between the stand and head of microscope like epi-fluorescence set.

Numerical Aperture (N.A.): This number is imprinted on the objective lens. It is a measure of the resolving power of the objective (how fine a detail can be seen). The condenser aperture diaphragm should be adjusted to the same value of the N.A. of the objective, to obtain the best results

LWD or ULWD: These abbreviations stand for “long working distance” or “ultra-long working distance”. These objectives are able to work with a large specimen-objective distance and are used for specific applications. Mainly on inverted microscopes but also other applications like metallurgical.

HOW TO BUY A MICROSCOPE

Why buy a Microscope?

A professional microscope is a tool to aid the pursuit of knowledge and an absolute necessity in many medical, scientific, agricultural and industrial careers. Children are encouraged to investigate and may turn to science careers. Others increase their understanding of the “worlds within worlds.” Through the microscope, “ordinary“ becomes extraordinary!.

Microscopy is usually an indoor experience.

INTRODUCTION

Five Microscope Selection Rules

Rule 1 – Don’t waste your money on junk!
Good microscope equipment just will not wear out in normal use. It provides a very satisfying educational experience for any user – child, science student or adult professional. The optics are of professional quality, the mechanical components operate smoothly and accurately, and the owner knows by its excellent performance that this instrument is not a toy.

Rule 2 – Never buy a microscope at a chain store, in a mall, or in a toy store!
With extremely rare exceptions, mall stores just do not carry quality microscope equipment, nor do chain stores or toy stores. Emphasis is on impulse sales and promotions with little regard for quality.
Science specialty stores and instrument dealers are local sources of good microscopes.

Rule 3 – Buy a microscope from a company that makes professional optics.

Rule 4 – Buy a microscope from a company that will not compromise quality on inexpensive instruments.
Optika has many types of microscopes. Even the inexpensive microscoscopes are of high technical quality.
The expense is saved by using time-proven designs – not by making frequent model changes requiring constant new tooling costs.
A professional optics company with a design department staffed by optical physicists and optical designers is the right kind of company to make your microscope – if the company applies its high standards to the less expensive microscopes it manufactures.

Rule 5 – Select a microscope supplier that offers a “Limited Lifetime Warranty” and carries the partsinventory to back up the warranty.
Proven designs, good materials and precision machining assure that a microscope has “the right stuff” to last a lifetime. Nothing gives stronger testimony to manufacturer’s confidence in the products than a “Limited Lifetime Warranty” against defects in materials or manufacture. Some very good optical companies have made sad compromises on less expensive microscopes and will not provide parts support for microscopes more than seven years old.

Select the right microscope for the age and experience of the user.

Buying a Microscope for a Child
Remember the cheap fishing rods and reels that chain stores would promote for children? Not only were they difficult to use, but the line would tangle and the reels would even fall apart! Thoughtful parents buy well designed “good stuff” that is not too complicated for their child to use and is ruggedly constructed of good materials. If children can master the complexities of computers, they can certainly learn to use a microscope.
Younger children will not benefit from the more demanding oil immersion technique required to magnify 1000x. They may also find a “monocular” easier to handle than a “binocular” microscope. Choose a compound monocular microscope with 4x, 10x, 40x objectives.
You know your child better than anyone does. Responsible teenage children can learn to use any instrument within your budget that is suited for an adult. They can learn oil immersion technique and the extra cleaning protocols that they must observe with regular use of oil on specimens. Their science teachers will encourage them in their quest for knowledge and give excellent “how-to” advice when asked.

Buying a Microscope for a Student or Hobbyist
Any microscope is capable of giving professional results. Refer to paragraphs on “Stereo Microscopes” and “Compound Microscopes” to determine which is right for you. If the answer is a “compound microscope,” there are many options listed in “Microscopes.”

Buying a Microscope for the Advanced User
We start with simple, but excellent, student microscopes that the user may upgrade at a later time. They are suitable for advanced college level science majors, doctors’ offices and specialized scientific use.

Frequently Asked Questions

What Can I see?
The first discovery is that just about everything that is alive or was alive really consists of smaller components called “cells.” “Simple” specimens take on an elegant complexity when viewed at the cellular level. A whole new universe of incredible beauty and shocking drama becomes visible through microscope lenses. Violence of the “cell eat cell” drama that exists in a drop of pond water can occupy an observer’s attention for hours.
With great magnification, we can observe life processes, see the cellular structure of our own blood, common bread mold, yeast that cause fermentation in beer and wine and cause bread to rise. Experts can identify the bacteria that makes us ill – and those that cause tooth decay, gum disease and plant diseases.
With special three-dimensional “stereo” microscopes we see how tiny multi-celled creatures move, the digestive tract of a worm, a fly’s eye, or examine fine engraving, collectors stamps, coins and jewelry.

What is a Microscope and How Does it Work?
A microscope is an instrument that “magnifies,” or makes objects appear larger in order to show detail that we cannot see with the unaided eye. Our own eyes cannot clearly see objects that are much closer than 12 inches. Microscopes solve that problem with curved lenses that bend light and allow us to examine objects and see cellular structure. At highest magnifications, the objective lens gets so close to the specimen it appears to be touching. The microscope eyepiece then compounds the magnification and projects the image on our eye.

How do I know what kind of microscope to select?
Different types of microscope magnify specific sizes and types of specimens. Select the type of microscope that will adequately magnify and also resolve the detail in the specimen you want to examine.

Can I take pictures through a microscope?
Saving microscope images has never been easier. Certain microscopes include “trinocular” heads or convenient optical tubes onto which the user may attach a video, digital, or photographic camera. Many viewers at one time can then share the image on a large-screen monitor, or the user can preserve as digital
computer files.

What is “Magnification?”
Magnification -- the microscope’s defining trait -- is also the most misunderstood. Usually stated in “diameters,” magnification is the mathematical product of multiplying the power of the “ocular,” or eyepiece, times the power of the “objective,” the lens closest to the object being examined. Microscopes with 10x eyepieces and three objectives of 4x, 10x and 40x, yield 40x, 100x, and 400x magnification, respectively. If a specimen has a true diameter of 1.0mm, at 40x magnification, the detail will appear as if it were 40mm in diameter.

What is "Resolution?”
“Resolution” separates a microscope from a magnifier. “Resolution” – the ability to discriminate between two close objects to distinguish detail. As The higher the “Resolution”, the closer two tiny points may be to one another and still be distinguished as two tiny points. Magnification without resolution simply “enlarges.”
Without the ability to discriminate more detail, it becomes “empty magnification.” Two close points will appear as one blurry blob when enlargement exceeds the capability of resolution. Theoretically limited by the
interaction of glass and light, true resolution varies according to the quality and design of the equipment and the skill of the user.

What is “Parcentered?”
Most microscopes have multiple “objectives” on a rotating nosepiece. As the user turns the nosepiece to bring another objective over the specimen, each image should be right in the center of the view through the oculars, with no change from one objective to another.

What is “Parfocal?”
As in the paragraph above, the user focuses on a specimen and decides to try a different magnification. When rotating a new objective into position, there should be only minor adjustment to bring the specimen into the same sharp focus as the previous magnification. This is “parfocality.”

TWO BASIC TYPES OF MICROSCOPES

We separate light microscopes into two basic classes, “Dissecting (“Stereo”) microscopes, and “Compound Microscopes.” There are two major distinctions between the two.

1. You will view your specimen in three dimensions with a stereo microscope, not so with compound microscopes.

2. Compound microscopes have enormous magnification range – from 10x to 1,500x, compared to practical limitations of 5x to 200x with stereo microscopes. Compound microscopes can reveal detail at the cellular level.

The Basics of Dissecting, or “Stereo” Microscopes
3-D Observation/Larger Specimens. Nothing beats seeing a specimen in 3-D! If you are working with larger specimens, or if you wish to use a microscope to inspect parts, plants, stamps, coins, insects, rocks, fossils or archaeological specimens, or to guide you during fine dissection, you need a “stereo” or dissecting microscope.
Stereo microscopes have the unique ability to see the third dimension – depth. This makes stereo microscopes the instruments of choice for surgeons, gemologists, electronic assemblers, denture makers and fine engravers, to name a few examples. Because two optical systems are joined on one frame, similar to binoculars, stereo microscopes must be perfectly “collimated” by the manufacturer, otherwise their use can be uncomfortable.


Stereo microscopes have “long working distance” objectives to enable larger specimens to be examined.

How Stereo Microscopes Give a 3-D Image:
The binocular viewer alone does not make a stereo microscope capable of 3-D. Each eyepiece must capture a separate image from its own magnifying lens, known as an “objective.” The paired objectives work together to show slightly different views of an object to obtain a three-dimensional image, the same way human eyes work together to obtain depth perception. The eyepiece multiplies, or “compounds,” the objective
magnification in the stereo microscope in the same manner as it does in the “compound microscope.”

Magnification and Stereo Microscopes
For technical reasons, the magnification capabilities of standard stereo microscopes are usually limited to much less than 200x – usually 10x-40x

Field of View and Stereo Microscopes
They may cover a field of view almost as large as a quarter.

Illumination and Stereo Microscopes
Dissecting microscope illuminator design commonly provides for “incident” light -- light falling on the specimen -- and “transillumination” – light passing through the specimen from a light source inside the base.

Special (optional) fiber optic illuminators offer very even incident illumination, and others provide very specific “spot” illumination.

The Basics of Compound Microscopes
Compound Microscopes/Smaller Specimens. This is the microscope preferred by medical laboratory technologists examining blood and urine, pathologists screening tissue specimens and smears for cancer cells and microbiologists identifying bacteria in cultures. If you are interested in examining biological specimens at the cellular level, you require a “compound microscope.” Compound microscopes take their name from the calculation of magnification by “compounding” the power of the eyepiece x the power of the objective to get total magnification.

How Compound Microscopes Work.
We have to start at the bottom, to describe properly how a compound microscope really works. Except for some applications in which light shines on the specimen from above, most compound microscopes use “transillumination” – light projected from below to pass through the specimen to the magnifying objective lens.
The magnified image of the specimen projects through the “draw-tube” to the “eyepiece” where it is magnified another 10x. The eyepiece sends the image to our eyes where the lens projects it on our retina. It is just like a 35mm slide in a projector, or a movie projector at the cinema.


How Compound Microscopes Work, a Little More Detail
1. The illuminator is usually beneath the specimen, which rests on a platform called a “stage”. A top the illuminator are lenses that help it transmit a cone of even, brilliant light onto another lens system called the “substage condenser.”

2. The “sub-stage condenser.” This device can be raised or lowered by gears to control the point of focus of the light it has gathered from the illuminator so that it focus sharply on the specimen.

3. The “iris diaphragm” The condenser is frequently equipped with a light valve called an “iris diaphragm.” The user may open or close this iris diaphragm to control contrast and sharpness.

4. The “objectives” Powerful magnifying lenses directly above the specimen. Although a microscope is an optical system dependent on several key components, it is the “high dry” objective (40x) and the “oil immersion” (100x) objectives that are essential for much of the microscope’s capability to magnify and resolve detail. The careful design and precision shaping of these lenses enable them to focus on the specimen so closely they may practically touch it. Our eyes normally cannot focus closer , but some objectives can sharply focus on the specimen within a distance equal to the thickness of a sheet of writing paper. The 10x “low dry” and the 4x “scanning objective” are helpful to locate an area of interest using their larger fields of view.

5. The “nosepiece” is a rotating turret that can hold multiple objectives. It must be of great precision to enable each objection to rotate into perfect position centered above the specimen.

6. Rising from the “illuminator base” is the microscope “arm.” This structural component holds the platform stage, the focusing mechanism, the drawtube and nosepiece, and at the top, it has a socket into which fits the “head” or “body” that holds the viewing optics. The condenser rack-and-pinion is attached below the stage.

7. Focusing mechanisms are usually one of two types. In one variation, the platform stage moves vertically with gears to bring it near or farther from the objective, which remains fixed in place. In the other type, the drawtube is moves vertically with the objective and nosepiece while the stage stays in place.

8. The viewing optics consist of the “head” and the “oculars,” or eyepieces. Binocular heads require prisms to divide the image-carrying light rays from the single objective to the two oculars. In most binocular heads, there is a comfort adjustment to bring the two eyepieces in line with distance between the user’s pupils. This is the interpupillary distance (IPD).

9. The binocular viewer also includes a “diopter adjustment.” This allows the focusing characteristics of each ocular to match the user’s own eyes.


Magnification and Compound Microscopes
Typical magnifications of compound microscopes may exceed 1000x. In the medical laboratory, the highest magnification most often used is 400x-500x, although a busy laboratory will frequently examine specimens at 1000x – 1,500x.

Field of View and Compound Microscopes
The field of view is tiny – as little as 1/5th of millimeter at 1000x magnification. About the largest field of you can expect with a compound microscope is 1/5th of a millimeter at 40x (using a 4x objective and a 10x ocular).

Software - OPTIVISION - QUICK MANUAL

DIGITAL CAMERAS & SOFTWARES

Resolution of cameras and digital microscopes

Optikam Pro 3 = 3.2 Mpixels ready for Vista (Vision Pro – calibration slide included)
Optikam Pro 5 = 5.0 Mpixels ready for Vista (Vision Pro – calibration slide included)
DM-5 (digital monocular microscope) = 480 K - ready for vista (OPMIAS software)
DM-10 (digital monocular microscope) = 480 K - ready for Vista (OPMIAS software)

DM-15 (digital binocular microscope) = 2.0 Mpixels ONLY ready for Vista (OPMIAS software 1.5) if software is 1.3 or 1.4 (old versions) = NOT compatible with Vista

DM-20 (digital binocular microscope) = 3.1 Mpixels ONLY ready for Vista (OPMIAS software 1.5) if software is 1.3 or 1.4 (old versions) = NOT compatible with Vista.

EMAMCAP

It is a free software used to record a video or preview an image on the PC screen. Do not use this software to capture photos.

OPTIKA VISION LITE (new from July 2010)

Capture an image
Lineal measure
Print report with logo (in BMP or JPEG format)
Live view with a bigger window
Save images step by step
Record a video-clip without switching to Emamcap
Keep in memory up to 20 different calibrations
Save a large number of measurements
New language in Polish

OPTIKA VISION PRO

Capture an image
Lineal measure
Print report with logo
Professional measure, more options of measurements. Possibility to export in excel file.
Create data base of measurements
Create a data base of camera settings per each end user personal vew of image.
Picture settings (change colors, gain, hue, etc....)
Manual cells counting.

IMAGE TOOL

Same as Vision Pro software but with automatic cells counting.

IMAGE J

Analisys software.

INSTALL SOFTWARE OPTIKA VISION LITE

1.- insert the CD into the CD device of your computer. The CD is an auto-run. (do not plug the camera in the USB port of your computer yet). In case the CD has problem to auto-run, please do the following:
Click on My PC
Click on CD Rom device
Click on Set up Optika

2.- you'll see 4 folders: drivers, emamcapture, manuals and vision lite.
3.- open vision lite folder
4.- select "setup.exe" to install software: vision lite.
5.- re-start computer
6.- plug in the camera into USB port of your computer
7.- your computer will show you a message "new hardware found" and will begin to install the camera's drivers

8.- if your computer shows a message "warning about missing XP certification.....", please click in NEXT and forget this step. (this message appears when the new hardware is not manufactured by Microsoft).
9.- end.

You have now the software and camera running in your computer.

How to set the camera in the microscope (monocular or binocular) or stereomicroscope

1.- remove the eyepiece from your microscope or stereo.
2.- insert the camera into the eyepiece-tube of your microscope or stereo.
3.- put a slide or object in the view path of your mciroscope or stereo.

I recommend:


In order to be fast to work with the software, we recommend to make a direct access : emamcap and VISION LITE in your computer's desk.

EMemamcap SOFTWARE

The emamcap is a software to make a pre-view image and record a video. Please do not use this software to capture photos. The emamcap is a help tool to center the object from the microscope through the view path of the camera and see the image centered in the screen of your computer before to take a photo with Optika Vision Lite software.

1.- double click on the emamcap icon. You will get a small window open.
2.- click on Devices.
3.- select driver of camera Optikam . You will get the image from the microscope+camera to your computer's screen. Sometimes takes a little time.
4.- focus the image.

Now the user has in focus the image from the microscope to the camera, he can go to Vision Lite software or:

Record a video. (AVI format)

1.- create a new folder in your computer where to store and save all videos.
2.- click on "capture" option from the menu bar.
3.- click on "setup" and you will see a new window open
4.- in the first tag, where says "video capture", please click on "capture to unallocated files", then click on browse and indicate the folder where do you want to store or save your future videos.
5.- in "video capture options", please indicate how long you want to record the video at "stop capture afer......." xxx seconds. Finally click in "o.k."
6.- close the set up window
7.- in the main window, now click again on "capture" from the menu bar
8.- click in "start capture". The software will stop to capture after xxxxx seconds.
9.- to view the video, please go to your folder (you have created in point number 1) and double click on the file you want to see.

Full screen

1.- click on "view" option from the menu bar
2.- click on "full screen"
3.- to get back just double click on the full screen.

Improve quality of image

1.- click "options" from the menu bar
2.- click on "video capture filter..."
3.- change contrast, brightness, gamma, etc....
4.- click on "apply"

OPTIKA VISION LITE SOFTWARE

Before to work with Optika Vision Lite, be sure you have closed the emamcap software.

Now open the Vision Lite software which has been installed in your computer at "start / programs / Optika / Optika Vision Lite".

Capture a photo

1.- indicate to the software the driver name of the camera you are using. Do following steps:

Ø click on the icon with a microscope to see on-line image in your computer.
Ø in the sub-menu please select driver of camera (for B1 camera, driver is TC131 USB CAM).

Ø re-focus image on your computer just by rotating the micrometric knob from your microscope. When focusing in the microscope, the image appear un-focused in the computer, that is produced by the difference focusing distances.
Ø to improve quality of image, got to settings.
Ø in the sub-menu, please click on "one push WB" to do auto-balance. Change other parameters if needed. Now you can click in "capture". The photo will be fixed on the screen.

To save a report document:

1.- in the left menu, please introduce a "title".
2.- operator: you can write your name
3.- notes: any note that can be of interest, for example the objective used to capture the photo 4x objective
4.- comments: introduce any comment that can be of interest.
5.- go to menu bar and click on "file"
6.- click on "save report as" , with this option you will save photo will all comments. Format to save it only in *.asa. The image can not be used with other softwares, only with Optika Vision Lite.

To save only photo:

1.- go to menu bar and click on "file"
2.- click on "export"
3.- click on "image to file"
4.- save picture only is format: bmp; jpeg or tiff

To open a report from our folder

1.- go to menu bar and click on "file"
2.- click on "open report"
3.- select a report to open

Import image from file

This option is used to download at Optika Vision Lite pictures saved in the past and view them at the main screen.

Calibration and measurements

It is important to calibrate the software before taking any measurement. For that reason, we the Optikam camera you will receive a measurement slide of 0,01mm

How to calibrate.

1.- put the calibration slide on the microscope stage and focuse it with through the camera until you see the lines. We recommend to work with 4x objective the first time.
2.- from the tool bar (icons) be sure that "active calibration" says 1. Empty
3.- from the menu bar, click on "tool" and then "calibration". Select "add new calibration".
4.- click "OK" on the small screen
5.- draw a single line from the left point until the right point and indicte 0,01
6.- enter measuring unit "milimeters"
7.- enter name of calibration, for example 4x objective
8.- choose in which position you want to save the calibration. (1. 2. 3. etc...)

Do same procedure from point 1 unitl 7 with rest of objectives 10x, 40x and 100x of your microscopes. When arriving to point no. 8 save each calibration in a different position.

Now you can do measurements. With Optika Vision Lite only is possible linear measurements.

If it is necessary user can export measurement to an excel file *.csv format

Print a report

1.- from the menu bar, click on "file" and then "print".
2.- from the tool bar, click on the icon of a printer machine.

You will obtain a printed page with picture and all measurements as well as comments and additional information.

INSTALL OPTIKA VISION PRO WITH OPTIKAM PRO 3 & PRO 5 - PROCEDURE

Note: do not plug the camera into USB port yet.

First of all, let's do install of software.

1.- Insert CD Rom into CD device of your computer. The CD is an auto-run. (do not plug the camera in the USB port of your computer yet). In case the CD has problem to auto-run, please do the following:
Click on My PC
Click on CD Rom device
Click on Set up Optika
2.- select language, next
3.- select camera, next
4.- select standard, next. You will see a small window that will inform you which driver and software will install. Please click on "accept"
5.- installation starts, click next
6.- introduce a user name
7.- introduce a company name
8.- select typical set up, next
9.- current settings, skip this and click in next
10.- if your computer shows a message "warning about missing XP certification.....", please click in NEXT and forget this step. (this message appears when the new software and hardware is not manufactured by Microsoft).
11.- click on finish

Second, let's install camera (hardware)

1.- plug in the camera to the USB port. You will see inmediatly a tag with "new hardware found", this is the assistant to install the camera. Please be sure you still have the CD Rom into the computer, and follow instructions on the screen.
2.- if your computer shows a message "warning about missing XP certification.....", please click in NEXT and forget this step. (this message appears when the new software and hardware is not manufactured by Microsoft).
3.- Finish

Third, creat a direct access of Optika Vision Pro in the desk of your computer

1.- click on start

2.- click on programs
3.- click on optika
4.- click on optika vision pro
5.- click with the right button of your mouse on optika vision pro and select from the sub-menu "create direct access to your desk"


OPTIKA VISION PRO SOFTWARE


Capture and save a photo

1.- put the sample or object on the stage of microscope
2.- put the camera on the third tube of microscope (be sure you have pulled out the pole from microscope head)
3.- you will see the image on the screen of your computer, this is an on-line picture.
4.- go to "view" from the menu bar
5.- click on "fit to window" for a better image.

Before to acquire the photo, please check quality of image. How to improve it:

Camera control panel

It is the menu of the left side of the window (vertical)

In this option user can improve in a fast way the quality in on-line image and save or store the values with a private name to download same values next time user is observing a similar object through the microscope.

Save values:

This option is used to save under a given name the camera settings, or calibration values or both at the same time.

Imagine many users need to work with the microscope but each of them work with different settings and calibrations values. Each user can save his/her settings with its own name. How to do it:

1.- focus the sample using microscope or stereo and see the image on-line at your PC.
2.- change the settings of the camera or do calibration.
3.- click on ADD and save these information with a name.

Camera control:

1.- click in autoexposure
2.- click on white balance

These options gets the best light. It is ALWAYS recommended to click on auto-exposure and white balance to get true colors of the object before to store the image.
Please do not do any photo yet.

3.- go to "capture options" from the vertical menu of the left.
4.- chek the "capture full field of view" is selected
5.- check the "preview captured image" is selected

Now do photo. You will see the photo in the screen.
6.- go to "file" from the menu bar
7.- click on "save as...." to store or save the picture with a name.

If you like to see all acquire photos in the vertical-menu before to save, please do the following.

1.- go to "capture options" from the vertical menu of the left.
2.- click on "field group"
3.- acquire a photo
4.- go to the top of the vertical menu and click on the green arrow (back)
5.- you will obtain all captured photos as a vertical list.


Time lapse:

This option is used to acquire a sequence of images. It needs to introduce 2 parameters:
1.- Interval : period of time between one picture and next picture
2.- duration: total time which is needed to stop working the sequence.
3.- the photos are shown in the main screen before to store or save.


Camera control - 2

This option is used to modify picture on-line to improve it quality before to take a photo.
1.- Exposure : is the exposure time of the camera. Using brightfield microscope, needs less exposure time. Using a epi-fluorescence microscope needs more exposure time.
2.- Gain: intensity channel of each pixel.
3.- Gamma: gamma rays of each pixel.
4.- Apply flat field correction: using only when we combine with different photos to obtain one flat photo.

Capture options:

Averaging - to reduce noise from picture. The best is number 8 usually used with epi-fluo or very dark images. Number 1 means no any reduction, to be used with bright samples.
Subsampling - helps to reduce even more the noise. To be used with epi-fluo samples.
To - where to store or save the resulting photo. ALWAYS select "file".
File - indicate name of folder and file name where to store the photo.
Field group - the photos are stored in a sub-menu before to save them definetely. To view the sub-menu go:

1.- go to the top for vertical left menu.
2.- click on the green arrow (back). It will show all photos positioned in vertical.

Camera control extended:

Use it to change the saturation, hue and brightness of image before to acquire a photo. This option is used to increase color intensity of the sample.

Measurement and Calibration:

Before to work on measurements, is important to calibrate the microscope and camera.


Calibration process :

1.- put the calibration slide on the microscope stage and focus it with the camera until you see the lines on the PC screen. We recommend working with 10x objective the first time.
2.- take a photo with best quality as possible (use camera control from the vertical menu if needed). For example with 10x objective (microscope) or 1x (stereo)
3.- go to measurement from the menu bar, click on “micrometer” from the sub-menu
4.- click on "calibrate" from the sub-menu
5.- draw a line from the left point to the end of right line and double click
6.- enter the following values:
*length of the line
*magnification (enter total magnification. Using the example of before this would be: 100x if microscope; 10x if stereo).
* unit
* square unit
* microns
7.- from the “camera control” left bar click at ADD. Introduce a name where this value will be kept. (taking the example, you can save with “objective 10x” if microscope or “objective 1x” if stereo)
8.- now, you have calibrated the equipment with 10x objective (or 1x if stereo). You must do all process from point 1 to 7 again to calibrate with each objective.

If you like to test the calibration after you have saved at the “camera control” bar each objective, please do the following:

1.- put the objective for example 4x if is a microscope (any other if is a stereo)
2.- see the image on-line on the screen of your PC.
3.- before to take a photo, please go to the “camera control” bar and select objective 4x (or any name you have given when calibrating).
4.- now take a photo.
5.- go to measurement and select a line. Draw a line over the sample you want to measure from the captured photo. The resulting values should be correct!!

Data base (bottom of screen) :

The data base is used to save and store pictures by author, date, short description inside the Vision Pro software. No need to create a new folder on the computer outside the Vision Pro software.

1.- take a photo
2.- click on "store to database"
3.- a new window will open, please introduce information.
4.- click in add. With the rest of icons of the data base, you can find a picture, eliminate, or load into the main screen.

To change color of the measurement lines:

1.- click on view from tool bar
2.- click on overlay color
3.- choose a color for he lines


To change the size of the measuring lines:

1.- click on view from tool bar
2.- click on overlay size

To change the size of text of measurements:

1.- click on view from tool bar
2.- click on overlay size

The database menu:

The best way to store and save a photo and a fast download is using the database menu. It saves all photos in the software outside the computer C: device.
In the database is easy to save a picture by name, date, author and a short description. All this information helps ot find it out in the future.
The picture can be saved, deleted or download from the data base.


The Field menu:

1.- Open: opens all photos saved under *.fov format
2.- Save: saves photos from the "field group" vertical menu under *.fov format
3.- import: import photos from different formats into the "field group" vertical menu.
4.- export: to save photos from the field group into a disk or external disk.
5.- append: to add the active image to the end of field group vertical menu. Also doing double click on the photo inmediatly puts it to the vertical menu.
6.- Load: to open a photo from the field group vertical menu. Also doing double click with the mouse
7.- Move up: to go to the previous photo from the vertical menu
8.- Move down: to go to the next photo from the vertical menu
9.- Clear: to delete the selected photo
10.- Clear all: to delete all photos from field group.
11.- Show previous: when all photos are active at the center of screen and want to see the previous photo.
12.- Show next: when all photos are active at the center of screen and want to see the next photo.
13.- Panorama landscape: to get a big picture horizontal from small photos.
ØClear all photos from the field group vertical menu.
ØTake photos from the slide or sample (as an example 3 photos from different points of the sample)
ØClick on panorama landscape to get the resulting photo.
14.- Panorama portrait: is the same as before but vertical.

15.- Multifocus composition: Mainly used in Stereozoom microscopes to get a resulting flat image. The stereozoom microscope gets images from different working distances because the object are tridimensional. Using the multifocus composition we can get a falt image. Also can be done with microscope, but is more difficult to appreciate the resulting photo.
16.- Vector arithmetics:
ØCombine: Combines the last 3 images from the field group vertical menu.
ØSplit: decompose colored image into 3 images representing GBR
Use these comands to make a comparison between different photos.

The Adjust menu:

This menu helps to improve quality of image on-line or captured photo.

1.- Gamma
2.- Image temperature: to set a new color temperature to be used at the white balance. Recomended to have always selected white color. This option only works on-line picture
3.- Area based white balance: to correct color aberration. This option only works on-line picture.
4.- Interactive : to change the hue, intensity and saturation.
5.- More : intensity of light, hue, saturation (also can be clicked directly from tool bar)
6.- Less: intensity of light, hue, saturation (also can be clicked directly from tool bar)
7.- Increments: to set the values of hue, intensity or saturation.
8.- Mirror: can be applied on both pictures: on-line or photo
9.- Rotate: only works with photo.
10.- Magnification: indicate to the software which objective has been used to capture the photo.

The Enhance menu:

This menu is to improve the quality of image. Use different filters to improve the image.
With on-line image only is possible to do the "Flatfield correction" and "spherical aberration correction".

With captured photo is possible to do the following improvements on the image:

1.- Flatfield correction.
2.- Denoise : to removo any noise.
3.- Remove bad pixels.
4.- Unsharp : to get sharp image.
5.- Max contrast
6.- Sketch
7.- Adaptive edges emphasis
8.- Equalization
9.- Amplitude depletion
10.- Darkfield : use this filter as a darkfield application on microscopy.
11.- Photometric transform: 2 options, a) create a black/white picture or b) negative picture
12.- Spherical aberration: to correct spherical aberration.


INSTALL FREEWARE SOFTWARES

1.- Insert CD Rom into CD device of computer.
2.- click on set up Optika
3.- select "Freeware"
4.- Select "personalized installation"
5.- From the menu of the window, select the softwared you want to use:
- GIMP - Photo retouching
- COMBINE Z - combines pictures to increase depth of focus
- IMAGE TOOL - image analysis, the most important cells / objects counting
- IMAGE J
6.- Follow instructions of the screen per each software installation. Most of the cases is clicking "next" or "yes" or "ok.".



* Combine ZM or Z5 (www.hadleyweb.pwp.blueyonder.co.uk)

This is a link to use a combine software, free !!!.

Software - OPTMIAS - QUICK MANUAL

This is a quick manual instructions for "first steps" to use OPTIMIAS from Optika

This software is supplied with DM (Digital Microscopes).
OPMIAS version 1.5 is now compatible with VISTA.
Other versions lower than 1.5 (like 1.2, 1.3 or 1.4) are NOT compatible with VISTA.

INSTALL OPMIAS - PROCEDURE

1.- Insert CD into CD Rom device of your computer and download OPMIAS software.

2.- Plug USB cable and locker (blue color) to the USB ports of your PC. It will identify both as “new hardware found”, please follow instructions from the screen to instal properly the drivers of camera and locker. Do not remove the CD rom from CD device of your computer.

3.- Double click on the icon of OPMIAS software to start with it.

4.- Go to help from the menu bar. A drop-down menu will appear, click to “local register”. Your locker and computer are now registered. “register success!”.

5.- Go to file from the menu bar, and select video-device. A small window will appear with the name of driver of camera: MC-D200.310V. Please select and OK.

6.- Go to file and select video capture. Also you can direct access by clicking on the icon drawn with video camera.
Capture window

Preview – see on-line sample from microscope.
Stop – stop on-line capture
Capture – take a picture. It is only possible to save it in bmp format
Auto-take – take pictures into a certain period of time. Introduce settings like: file name,
time and frames/second.
Record – record a video only is possible with avi format.
V format – change the image sequence. This option is used to modify the speed of
images transmittion from the microscope to PC and resolution.
V source – to modify settings of camera to get better quality of image: contrast, hue
(background), brightness, exposure, etd…
RGB – to modify red, green and blue on the image.

Click on return to go back to the main screen.

Edit Menu

Copy - copy into clip board the picture.
Paste- paste what is inside clip-board.
Paste selection – paste what is inside clip-board.
Clear clip boar – delete what is inside clip-board.
Resize- modify size of picture.
Delete image

View Menu

The view menu shows or hide the bars of the software.
- tools
- status
- measure
- graphics
- control
- normal screen
- zoom in
- zoom out
- full screen
- magnifier: when click on this option a magnifier draw appears, just click on the image and move the mouse over the photo.


Process Menu

Flip vertical or horizontal.
Rotates image.
Insert filters like: blue, noise median or equal, sharp.
Edge, edge horizontal or vertical.
Split RGB: eliminate colors to obtain a picture of grey scale, a picture without blue, a picture without red and a picture without green. All images can be seen at the control bar menu at the right side of screen.

Adjust Menu

To apply more filters over the captured image, like negative, gray or gray scale, adjust in: brightness, contrast, gamma, high bright, middle bright, etc…..

Calibration

1.- Put a calibration slide to the microscope stage and take a picture (use 10x objective for best quality of image)

2.- click on “measure” from tool bar. A drop-down menu will appear.

3.- click on “calibrate special measure”.

4.- from window “calibrate measure” select the unit. As a sample, let’s calibrate in micron. Click on “micron” button.

5.- click on “calibrate” button

6.- draw a line from start to end (1 micron length). Introduce number 1 at the “actual value”.

7.- click on “save” button and save the file with a name, for example: calibration test 1 micron. Click accept.

Test measurement is correct

1.- click “load” button.

2.- choose the file you want to test, for example “calibration test 1 micron”.

3.- click “test” button.

4.- draw a line to measure
5.- chech at “test result” the value, it should be correct (for our example it is 1).

Mesure window

Shows a table where to save value of measurements.

Merge visible

Put a copy of the photo with measurement at the control bar on the right side of the screen.

Measure

By clicking to this option, there’s drop-down menu to choose the type of measurement.

Tag

To highlight in a particular place on the photo. It is also possible to insert a short comment a the table.

Count

Just counts total points.

Icons from the “Tools bar”

Image operation – combines 2 images into 1 resulting image, it is like an “amalgamation”. Important: both images must have same size.

Merge image – combines 4 images as a puzzle.