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Focus Drive Manual


This page describes a microscope focus drive, or z-stage, for a Meiji Techno EMZ stereo microscope. The  focus drive allows precise, computer-controlled, vertical positioning of the microscope. This is useful when taking photographs through a microscope.
This page guides you through making an enhanced depth-of-field picture of a 10mm high object. A separate page explains how to use the focus drive with Micro-Manager microscopy software.


Stereo Microscope

A Meiji Techno EMZ5-TR stereo zoom microscope with focus drive
A Meiji Techno EMZ5-TR stereo zoom microscope with focus drive.

 

If you take a look through a stereo microscope, you immediately spot the difference between a stereo microscope and an ordinary, "biological" microscope. The image seen through an ordinary microscope is flat, as when looking a photograph. A stereo microscope shows a three-dimensional image. The image has depth, which allows you to manipulate tools under a stereo microscope; for instance to assemble products.


Magnification


What magnification should one expect from a stereo microscope? Stereo microscopes have lower magnification than normal ("biological") microscopes. Stereo microscope magnification is generally between x2 and x100. An example may help. If you look at your wristwatch with the naked eye, you can see the second hand move. If you look at your wristwatch using a stereo microscope, you can see the minute hand move. Finally, the magnification of a biological microscope would be sufficient to see the hour hand move.


Photography


The depth of field of the microscope image is small - a few millimeters at best, a fraction of a millimeter at worst, depending upon the magnification used. This means that if you takes a picture of a 10mm (approx. 0.5") tall object, typically either the bottom or the top of the object will be in focus, but not both at the same time.
To overcome this problem, many different pictures are taken, each focused at a slightly different height. These pictures are then joined into a single picture with increased depth of field using stacking software such as Helicon Focus.  A few sample pictures show how this process may improve your pictures.
Taking a large number of pictures manually can be very time-consuming. If you wish to automate taking these pictures, you need computer control of both the camera and the microscope.


Tethered shooting


When taking photographs through a microscope, the camera is usually connected to a PC. Using a PC to control a camera is called tethered shooting. The PC screen is used as viewfinder. Camera settings such as focus, aperture and shutter time are adjusted on the computer, and photographs are captured directly on the hard disk.
There is a vast choice in tethering software. PSRemote from Breeze Systems is a good example. PSRemote provides both a Windows and a command-line version. The Windows version can be used interactively; while the command-line version is more appropriate for batch scripts. Once the tethering software is installed and configured, taking a picture and saving it to disk becomes child's play:

C:\zstage>PSRemoteTest.exe
Success, image saved as: C:\PSRemote\2010-09-24\0001.JPG

Other camera models may need other software, but the basic idea is the same. You connect the camera to a computer and take pictures under software control.


Focus Drive

Installing


We need to connect the focus drive to the computer, and install the focus drive software.
Connect the focus drive to a PC serial port or a USB-to-serial adapter. Connect the focus drive to the 24V power supply.
Download and install the command-line utilities for Windows from the Downloads section at the bottom of the page.


Using


A focus drive allows the computer to change the vertical position of the microscope. The command-line utility zstage.exe is used to move the focus drive. zstage.exe takes two parameters: the serial port the focus drive is connected to, and the distance the focus drive has to move. The output of zstage.exe  is the current position of the focus drive. An example: the focus drive is connected to port COM3, and we wish to know its current position:

C:\zstage>zstage.exe COM3 0
.
position 0
C:\zstage>

The focus drive reports back its current position as 0.
Now let's move the microscope. Distance is specified as number of steps * 256. One step equals 19.25µm. Let's try to move the microscope one step down:


C:\zstage>zstage.exe COM3 256
.
position 256

The focus drive reports back its current position as 256. If we move one more step down, position will be 512:


C:\zstage>zstage.exe COM3 256
.
position 512

Let's go back to the origin:


C:\zstage>zstage.exe COM3 -512
.
position 0

C:\zstage>

Drive behavior can be summarized as follows:

Microstepping improves resolution at the cost of decreased repeatability. For most applications, microstepping is not needed. In most cases, specifying distances which are a multiple of 256 will  provide the best results.


Depth of field


How many pictures do you need to take? How much should the microscope move between pictures? A bit of experimenting usually is needed, and a bit of experience helps, but where do you begin?
As a first guess of how many pictures will be needed to show an object in focus, look up the depth of field in the table "Depth of Field of the EMZ5". Set the vertical distance between two pictures to half the depth of field, rounding down. Set the number of pictures to the height of the object divided by the vertical distance between pictures, rounding up.


Example:


Question: As a rough guess, how many pictures are needed for a 5mm high object, with the Meiji Techno EMZ-5 at 2x zoom, and without auxiliary lens? The focus drive has a vertical movement of 19.25µ per step.

Calculating the number of pictures
Object height: 5 mm (measured)
Depth of field: 0.391mm (look up in EMZ5 table)
Vertical distance between pictures, in mm: 0.391mm/2 = 0.196mm (depth of field/2)
Focus drive step size: 0.01925mm/step (from focus drive documentation)
Vertical distance between pictures, in steps: 0.196mm/0.01925mm/step = 10.18steps
Rounding down: 10 steps = 0.1925mm between pictures
Number of pictures: 5mm/0.1925mm = 26.0 pictures (object height/vertical distance between pictures)
Rounding up: 27 pictures

Answer: 27 pictures, moving the microscope 0.1925mm = 10 focus drive steps between pictures. This corresponds to a 'distance' parameter of 10 * 256 = 2560 for zstage.exe. Assuming the focus drive is connected to COM3, the following command will be used to move the stage:

zstage.exe COM3 2560

Taking the pictures


We now combine everything in  a batch script.

script parameters
number of pictures: 27
command to take a picture: PSRemoteTest.exe
vertical distance between two pictures: 2560
command to move the stage: zstage.exe COM3 2560

We are now ready to take the pictures. Manually focus the microscope to the bottom of the object. Executing the batch file zstagerun produces this sample output. During execution of the script, a total of 27 pictures are saved in C:\PSRemote. Check the pictures are OK. If the color of the pictures is off, verify the color temperature setting of the camera corresponds to the light source you're using. All pictures of the stack should be as similar as possible. If autofocus or auto-exposure causes variation between pictures, you may have to use manual focus and/or manual exposure.


Z-Stacking


The pictures can now be processed with stacking software such as Helicon Focus . This is an operation which requires a fair bit of computing power. The end result is a single image with extended depth of field.
At this point you may wish to experiment. Can you take less pictures and still have the same image quality? Does increasing the number of pictures improve image?  Changing the parameters in the zstagerun batch file provides the answers to these questions in minutes.


Resolution and depth-of-field of the Meiji Techno EMZ-5


Photo Focus Drive EMZ Front
Numerical Aperture and Resolution table of EMZ-5 Zoom Stereo Microscope
Zoom Magnification N.A. Resolution power
Standard With Auxiliary 0.5X With Auxiliary 0.75X With Auxiliary 1.5X With Auxiliary 2.0X
0.7X 0.027 12.4µ 24.9µ 16.6µ 8.3µ 6.2µ
1X 0.039 8.6µ 17.2µ 11.5µ 5.7µ 4.3µ
2X 0.056 6.0µ 12.0µ 8.0µ 4.0µ 3.0µ
3X 0.064 5.2µ 10.5µ 7.0µ 3.5µ 2.6µ
4X 0.067 5.0µ 10.0µ 6.7µ 3.3µ 2.5µ
4.5X 0.07 4.8µ 9.6µ 6.4µ 3.2µ 2.4µ

Depth of Field of EMZ-5 Zoom Stereo Microscope with Standard SWF10X Eyepieces
Zoom Magnification
 Depth of Field in millimeter (mm) with SWF10X
Standard With Auxiliary 0.5X With Auxiliary 0.75X With Auxiliary 1.5X With Auxiliary 2.0X
0.7X 2.176 5.107 3.069 1.367 0.994
1X 1.053 2.467 1.484 0.662 0.481
2X 0.391 0.958 0.561 0.241 0.174
3X 0.244 0.623 0.355 0.148 0.105
4X 0.188 0.499 0.278 0.112 0.079
4.5X 0.164 0.44 0.244 0.097 0.068

Vertical Resolution of the Focus Drive

Used with the Meiji Techno FC ("Fine/Coarse") focus block the focus drive has the following mechanical resolution:


Connectors

The front panel of the focus drive has three connectors: power, data and the optional joystick.

Focus Drive Front Panel with Connectors 
Focus drive front panel with connectors.

Bottom: Power

Power connector

24V DC@0.5A, center pin positive. Do not exceed 30V. Do not apply reverse polarity.

Middle: Data

Serial port

Serial data, 9600 8N1.

Pinout
Signal Pin
TxD 2
RxD 3
Gnd 7

Top: Joystick

Joystick connector

Connects optional joystick for standalone use. Always unplug the power connector before connecting or disconnecting the joystick.

Pinout
Signal Pin
+5V 1
Digital out (GPO) 2
Analogue in (GPI) 3
Gnd 4


Links

Focus Drive

Tethering Software

Focus Stacking Software

Microscopy

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