Specifications

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MetaMorph Imaging Handbook Update 6/4/13

Startup

FIRST turn on mercury lamp (Fluorescence)

Computer and monitor

Qimaging Camera (on top)

Uniblitz Shutters-2

Halogen Lamp (Transmitted Light)

Computer

Domain is Cornell

MetaMorph options

MetaMorph- normal use

Offline-computer only

Check out microscope

Light path to correct camera vs eyepieces

Mechanical shutter open

Zoom set to 1

Filter turret – check filters installed, change if necessary

DIC filters and ND filter out

Objectives: dry: 4x, 10x, 20x,

oil: 40x, 60x, 100x,

H2O: 40x w/ cover slip, 40x dipping

Iris diaphragm on 100X oil obj - open

Main Taskbar

If you lose the Main taskbar, go to

Journal –Taskbars-Load Taskbar

Look in C:\MM\Taskbars\Main.JTB

Shutters

Check shutters using shutter buttons in Main Taskbar

Make sure shutter is set to NC = Normally Closed on shutter control box

You may have to hit the Reset button on the shutter control box

General Procedure

Find your sample and focus

Send light to camera by pulling out lower camera knob on right all the way

In Taskbar:

Choose QImaging camera

Acquire window:

Choose Setting- select correct shutter

Select exposure time or try Autoexpose

Hit Show Live

Adj. focus, exposure time and/or 12 bit grey scale

Unclick Saturation warnings if not wanted (may have to click 2x)

Acquire (shutter will open and close)

Save As (from Main Taskbar)

HELP

Use the F1 key to get help on any

active menu.

Choose Dialog for an explanation of

all entries.

MetaMorph help.pdf on website

Shutdown

Check Calendar before shutting down

Exit Metamorph

Leave Main Taskbar open

Transfer files

Logoff Windows

Turn off camera (on top)

Turn off shutters(2)

Turn off monitor

Turn off halogen lamp

Turn off mercury lamp last

Wipe oil off objective with lens paper

Cover microscope

*Note that Hg lamp must cool for 15

min before restarting

Summary of content (13 pages)

PAGE 1
MetaMorph Imaging Handbook Update 6/4/13 Startup FIRST turn on mercury lamp (Fluorescence) Computer and monitor Qimaging Camera (on top) Uniblitz Shutters-2 Halogen Lamp (Transmitted Light) Computer Login net id and netid password Domain is Cornell MetaMorph options MetaMorph- normal use Offline-computer only Check out microscope Light path to correct camera vs eyepieces Mechanical shutter open Zoom set to 1 Filter turret – check filters installed, change if necessary DIC filters and ND filter out Objec
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Acquire (Main Taskbar) Settings (lower left) Select fluorescence or transmitted. This chooses the shutter as well as exposure time, 12 bit adjustment, binning, gain, offset, etc. You can save your settings with the same name or a new name. Display Tab Spread Triangles to ends, adjust as needed. Right triangle makes pixels whiter, left makes background blacker. Middle is gamma, keep at 1 Special Tab The Qimaging camera has a gain, offset and speed control. Gain ranges from 1 to 30.
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Image Processing 12 bit Images Collecting images with 12 bits (4096 gray levels) is useful when you are doing quantitative work, complex image processing, especially involving thresholding, or when you have very low light levels or high background. Adjusting 12-bit images Use the triangles (under Display in the Acquire window) to adjust the white and black levels. (This is just like Photoshop using Image-Adjustment-Levels.
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Save Partial, to save just the defined region without re-acquiring To use this region again, use acquire active region on image. Overlay Images (Main Taskbar) Collect images sequentially using different filters or transmitted light Choose # images = 4 or more Choose an image for each color (use black for transmitted light image) Click on Show preview and move preview box around.
PAGE 5
Image Information File-Open, highlight any file name, will give exposure time, bit depth, image size, magnification, binning, gain, offset, speed, etc. OR— Open image Edit menu –Image Info—gives above info and more. Click on any file to see thumbnail image. Not with 12-bit images. Saving and Naming Images Images save in TIFF format. The .tif. is added automatically. Do not compress your images. Opening and closing compressed images causes loss of information over time.
PAGE 6
Transmitted Light (Bright Field) Introduction You can collect a bright field image of the same region as the fluorescent image to aid in localization of your fluorescence. The bright field image can be overlaid (merged) with the fluorescent image(s), see Image Processing. Note: Adjusting the microscope for an optimal bright field image is more complicated than fluorescence. It is highly recommended that you ask for help the first few times.
PAGE 7
Image Analysis -- Measure Menu Updated for new software. This is a brief overview. There are lots of things not mentioned here. Defining Regions Define region(s) with box or other drawing tool from Region Toolbar. User Traced Line to click-draw any shape See Region Tool Properties (last icon on Region Toolbar) for other settings To delete active region, hit del key. You can shrink, save, move, transfer, label, and color regions.
PAGE 8
Hit Measure (at bottom) Objects measured will be green with white border. Border is an option under Preferences Object Data—display of data Click on any data point to highlight object on image or vice versa Double click to remove any data point/object Data Log is in this view Reset Current to delete measurements and start over. Filters Use a filter if you want to limit measurements to objects of a certain size range or grey value range.
PAGE 9
Collecting a Z-Series Set Up Attach Z-motor color to coarse focus of microscope Must start MM with the Z-motor icon Use Show Live in the Acquire window to determine the best exposure for entire series Use a bright plane in your specimen Insert and tighten focus motor sleeve on right focus knob. Push in while tightening screw. Make very snug. All focusing must now be done with the small knob next to the joystick.
PAGE 10
Timelapse Imaging – Basic Choose in Main Taskbar For transmitted light or fastest times, use none for shutter Interval—shortest is ~400msec, accurately Uncheck time reporting Set time or number of frames There is a maximum file size based on RAM of about 10 min at 0.5msec To go faster or longer Uncheck viewing Use center quad Use binning Use 0 Interval, but times may be uneven (you can get the values) Save as .
PAGE 11
PhotoShop (ver 5.5 or 6.0 of 7.0) with Metamorph Enhancing Contrast or Adjusting Colors Image--Adjustment--Levels choose RGB or individual color use the histogram to adjust brightness, black level, and gamma After adjusting, you should save with a new name. The contrast changes you make are permanent and if you don’t like the printout, you will want to start with the original image, not the adjusted one. Always save the original image.
PAGE 12
Specifications Microscope: Olympus BX50 Camera: QImaging Retiga Exi cooled CCD camera 1392 x 1040 pixels Software: Metamorph Premier ver 7.7.5 from Molecular Devices, Inc.
PAGE 13
Scale Factors for Qimaging Retiga Camera with MetaMorph Obj Zoom um/ pixel pixels/ um Mag on screen Obj Zoom um/ pixel pixels/u m Mag on screen 100x 1 0.10 9.8 2800x 20x 1 0.52 1.94 585x 100x 1.25 0.08 12.28 3500x 20x 1.25 0.41 2.43 730x 100x 1.6 0.06 15.76 4480x 20x 1.6 0.32 3.12 935x 100x 2 0.05 19.8 5600x 20x 2 0.26 3.9 1170x 60x 1 0.17 5.85 1680x 10x 1 1.03 0.97 280x 60x 1.25 0.14 7.32 2100x 10x 1.25 0.83 1.21 350x 60x 1.6 0.11 9.