Blizard Advanced Light Microscopy club Making friends with your microscope

Your eye – the microscope Specimin Lens Camera/ Retina Image Processing Image is formed

Inside a microscope Eye piece:has a 10x lens in generally to increase magnification Tube lens: gathers light from the eyepiece And focuses them on the eyepiece Objective: gathers the light passing through Or emitted from the specimin Condensor: gathers the light from the light source and focuses it onto the specimen. The condensor can have special accessories to alter the type of light coming through Such as phase rings for phase contrast microscopy

Objective Table 1

Contrast versus resolution Contrast is the difference between the brightness of various details in the object. An 8 bit computer image has pixels in 256 shades of gray, ranging from intense white (256) to intense black (0). The "gray-value" of the pixels lies between 0 and 255. Resolution is the finest detail actually visible in the image (the resolving power is the highest resolution possible with the lenses in question). Contrast Resolution High Low

Resolution 2 The objective sets the ultimate resolution limit of the microscope. Resolution is affected by: The wavelength of light used to illuminate the specimen, Angular aperture of the light cone captured by the objective, The refractive index in the object space between the objective front lens and the specimen. (oil versus air or water) Resolution = l/2n(sin(  )) where, l is the illumination wavelength, n is the imaging medium,  is one-half of the objective angular aperture The numeric aperture of your lens is defined by n(sin(  )

To oil or not to oil? Refraction Objective (Numerical Aperture) Resolution Limit (um) Projected Size (um) 1x (0.04) air6.9 20x (0.5) air x (0.75) air x (1.30) oil x (1.40) oil By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness of the cover glass are reduced because oblique rays of light no longer refract

Spherical abberation Spherical aberration- focusing rays more tightly if they enter it far from the optic axis than if they enter closer to the axis. It therefore does not produce a perfect focal point.

Chromatic abberation chromatic aberration (also called achromatism) is the failure of a lens to focus all colors to the same pointlensfocuscolors

Field curvature Curvature of field in optical microscopy is a common and annoying aberration that is familiar to most experienced microscopist

Correction for abberation Objective Type Spherical Aberration Chromatic Aberration Field Curvature Achromat1 Color2 ColorsNo Plan Achromat1 Color2 ColorsYes Fluorite2-3 Colors No Plan Fluorite3-4 Colors2-4 ColorsYes Plan Apochromat 3-4 Colors4-5 ColorsYes

Condensor Gathers the light from the light source and focuses it onto the specimen Dr. August Köhler in the late 1800s devised a method for focussing all light beams on a specimin. This is called Kohler illumination and is standard in microscopy today

Setting up Kohler illumination First The lamp filament is focussed on to the focal plane of the condenser aperture diaphragm using the collector lens. 1 Close light on the Condensors aperture diaphragm You will see Something like this Focus light Coming onto The condensor Using 1.

Setting up Kohler illumination -2 As the sliders are opened and closed the path of ray traces through the condenser lens system. This influences the effective working numerical aperture of specimen illumination Centre the illumination Use Field Diaphragm Diameter and Aperture Diaphragm Diameter sliders to adjust amount of light let though.

Common problems Your image looks fuzzy at the edges – The Kohler illumination isn’t aligned properly, you need to reset it. Your image is in focus in the middle but not at all of the edges – Probably field curvature. Clean the lens as its not performing properly. Only the top or bottom part of your image is in focus. Your sample isn’t flat. Check the slider holder, is it in properly? Did you leave your samples completely flat when you mounted them. Can’t see your specimin? Maybe one of the shutters is closed or the light is going to the camera.

Eyepiece and camera – looking at your sample Both your eye and the camera collect the image Your eye is a lot more sensitive than the camera In fluorescent microscopy the camera only detects the intensity of light and not its colour. In histology microscopy the camera collects the intensity of Red Green and Blue light and combines them to give your colour image

Your Camera / detector To visualise your data and record it the light is projected onto a camera or a detector.

How the light moves onto the CCD chip 1. Number of pixels on a chip determine how big a field of view the camera sees. Its never as much as you see by eye 2. The size of the pixels determine the resolution of the camera. The bigger the pixels the worse the resolution x 1024 pixels, 8 bit image CCD camera chip Cameras collect data on a chip. The chip is composed of a certain number of pixels (mini detectors).

Contrast on the camera Cameras will often allow you to choose how much contrast detail you have. 8 bit = 256 levels of contrast 12 bit = 4095 levels of contrast 16 bit = levels of contrast You can find out how much contrast you have by checking the histogram Bad Good Intensity Frequency Increasing the amount of light on the camera by making your exposure time longer helps resolve poor contrast problems

Binning You can get better contrast sometimes by increasing the chip size using binning. This combines the signal from 4 (2x2) or 16 (4x4) chips but you lose resolution CCD camera chip No binning CCD camera chip 2x2 binning

Saving your data Tiff – Good as it keeps all the contrast and resolution information in your data. But the file size is large. JPEG – Not great as it averages the contrast and resolution when it saves your data, the more often you save the more the information is averaged. But file size is small Format made by the manufacturer. BEWARE! Manufacturers have a nasty tendancy to update their software. e.g. Zen, new Leica software. Your old files will often not open at all or correctly in the new software. Save your data in a non-proprietorial format! especially if you are a career scientist.