If you are working with specimens in brightfield or transmission mode, then you can use most any camera. The less expensive, analog cameras will have more noise, however, and create images that will not deconvolve quite as well as images captured with digital cameras. The instances in which VayTek recommends an analog camera are limited to users who have small budgets, who need the analog camera for live recording, and who will be doing only occasional deconvolutions, usually for improving images for publication. Images captured with analog cameras are confined to the nearest neighbor algorithm. Deconvolution is really designed for fluorescent specimens. This is because the mathematical formulae for the deconvolution algorithms assume the light in the specimens radiates equally in all directions. Consequently, the implications of specimen characteristics for choosing a camera become more obvious. If you are working with very bright fluorescent specimens, then you may still be able to work with an analog camera, although the analog camera will not give the best results for deconvolution. A digital camera will be a much better choice, even for bright specimens. There are several reasons for this. Digital cameras have better dynamic ranges than analog cameras - more gray levels mean more accurate deconvolution. Digital cameras, which are usually cooled, have lower noise and are better for deconvolution - the mathematics of deconvolution will amplify noise in an image so it is important to minimize it during acquisition. Digital cameras integrate over time, thereby letting you work with a wide variety of fluorescent dyes and intensities of dyes. And finally, digital cameras usually have more pixels on the chip and yield images with higher resolution, often at the resolution limits of the microscope. If you are planning to conduct experiments in which you will be measuring the intensity values of the pixels in the image, you will need to use the constrained iterative algorithm for deconvolution, probably with a measured point spread function. This means you will most certainly need a cooled, digital camera for data acquisition.

Appropriate Applications:

Broadcast-resolution CCD cameras are not suitable for applications such as chemiluminescence, fluorescence, and confocal microscopy. Scientific-grade CCD sensors provide better spatial resolution and higher image fidelity, thanks to better quantum efficiency, lower readout noise, and lower dark count (see Dark Current definition).

Binning Control:

Pixel binning is a method whereby adjacent pixels are added together to form a superpixel. This is usually accomplished through software control. Pixel binning increases the sensitivity of the imager, and the signal to noise ratio will increase because the effects of individual photons or electrons will be reduced. The practical result for the user is reduced integration time. However, there is a trade-off between increased sensitivity and lower resolution. Binning is a good method for focusing. For an example of binning options, ROI, and frame rates (frames per second) see the Cooke Camera chart.

Chip Bit Depth:

There is considerable confusion as to the meaning of bit depth and noise. All chips have a bit depth rating from the manufacturer. For example the Sony interline and the Kodak KAF chips are both 12 bit chips. However, the effective bit depth of an image from these two chips is quite different. The Sony chip has a well depth of about 18,000 electrons. Given that it has a readout noise of about 9 electrons per well, the effective signal-to-noise ratio is about 18,000/9 or 2,000. This means the camera can produce an image with about 2000 shades of gray - or an image with a true bit depth of about 11 bits - not the rated 12 bits. On the other hand, the Kodak chip has a well depth of 40,000 electrons and a read noise of about 10 electrons. This gives it a true bit depth of 40,000/10 = 4000 or 12 bits. Visually, the images from these two chips are very similar. However, if you perform an exacting deconvolution process on images from these two cameras, you will see slight differences in the results. This is even more obvious if you are working with dim specimens and the dark current noise builds up over time and adds to the readout noise. If you are working with very dim specimens, it is better to select a camera with a higher well depth, even if it means sacrificing readout speed.

Chip Grade:

Chip grade manufacturers grade their chips to indicate how free they are from defects such as dead or hot pixels. The best chips are the most expensive and are usually called Grade 0, or Grade 1, depending on the manufacturer. Some camera makers will create their own grading system in a game of "specmanship". For example, they will supply a grade 2 chip from the manufacturer but call it a grade 1 using their own grading scheme. The camera maker will specify their grades as going from 0 to 2, while the chip manufacturer's grading scheme goes from 1 to 3. Make certain that you ask about the chip grade and if it is the camera company's grade or the manufacturer's grade. Also, ask about the range of grades so that you will know where your camera fits into the overall scheme.

Chip Size:

The overall size of the chip as measured diagonally. The pixel size is in microns.

Chip Type:

Some cameras have a QE chip is a Quantum Efficient chip. It can be used instead of the standard chip. It lets the user integrate for 20 to 40% less time. Most of the camera manufacturers are incorporating QE chips as an option in their cameras. This raises the price of the camera $3000 to $4000.

Color yes/no:

Defines the camera as a monochrome camera, a color camera, both, or a monochrome camera that can be used with a color adapter, like the CRI filter.

Color Acquisition Method:

Color cameras create color images in four different ways:

with a mechanical color filter wheel
with an electronic filter (e.g., acousto-optic)
by masking a chip and using a Bayer filter
using three separate chips, one for each red, green, blue channel.

All color cameras with filters use broadband filters for red, green and blue. On the other hand, a microscope with a monochrome camera and a filter wheel captures color images that are more accurate because they use a filter wheel with the glass filters designed specifically for the wavelengths of your specimen dyes.

If you use a color camera to collect color fluorescent images you are adding additional filters in the optical path, which causes some additional loss of light and increases integration time. The exact amount of the loss depends on which color camera is used. For more information, including graphs comparing filters, see http://www.omegafilters.com/

The color cameras generally take longer to capture three separate channels and merge them into a single color image than does an excitation filter wheel and 12 bit monochrome camera.

And finally, the color cameras are not as well adapted for precise fluorescent imaging and some of the demanding results required from color images. For example, the color cameras with mechanical filter wheels can introduce vibration into the camera and cause slight misalignment in the color channels. The Bayer filter approach results in a loss of resolution. The acousto-optic filter reduces light transmission by about 30%.

In short, for the purpose of deconvolution, you will have more control of the acquisition process, better control and more accurate images with a good monochrome camera and an excitation filter wheel.

Color Tradeoffs:

See above. There are three basic ways to get color images from a black/white camera chip: 1) a Bayer filter, 2) the use of one chip and filters (glass, acousto-optic, or dichroic), 3) by using three chips with filters. 1) The Bayer filter places a mask on the chip and reserves subregions for each color. The light is split between these regions and combined to produce a color image. This method is fast and cheap, but the images are smaller and fuzzier. 2) The second method filters the light and snaps three images: red, green and blue components. This results in really good quality images, but the method requires three times as long compared to other methods, plus separate integration times for each color. 3) The three-chip method is more expensive, but is faster than method #2.

Coolest Temperature:

The lowest temperature achieved by the camera. The lower the temperature, the less noise. Cooling temperature only becomes relevant for longer integration times. It is important to note whether the temperature is reported as absolute or below ambient. For example, -20 C below ambient is not the same as -20 C absolute. If ambient temperature is 20 C and the camera cools -20 degrees below ambient, then the camera temperature is 0 C, and NOT -20 C. Some sales people don't know this distinction and can easily give you the wrong information. Be sure to check the official specification sheets for this value. VayTek's camera comparison database clearly shows the distinction between below-ambient and absolute temperature specifications. See page 3 and page 4 of the camera comparison database.

Cooling Process:

Peltier or thermoelectric coolers are common in scientific CCD cameras. The cooling devices are solid-state heat pumps that transfer heat from one side of the device to the other when electrical current is applied. The amount of heat reduction achieved varies. The temperature differential between the two sides lowers the dark current of the CCD sensor dramatically. See "Dark Current" definition above.

Comparison Images:

Using standardized capture conditions, VayTek has produced a set of comparison images for some of the most popular cameras for digital imaging systems for science, medicine, research and microscopy. The capture conditions were:

40x .75NA lens
Olympus BX60 microscope
Mercury lamp
500 ms exposure time for all images
A new part of the specimen was used for each capture to avoid bleaching out the specimen
The specimen was a triple stained slide prepared by Molecular Probes. It was a commercially purchased stain: Bovine pulmonary artery enothelial cells, fitc, dapi, texas red; green is tubulin, red is F-actin, and blue are nuclei.
To view the Comparison Images, click here.

Connectors on Housing, Number of:

Relevant for users who want to minimize wiring nests. More connectors indicates more user-controllable features.

Dark Current:

Impurities in a CCD's silicon layer cause some pixels to suffer from dark current more than others. These pixels build up thermal electrons at a faster rate than the rest of the pixels in the CCD. To reduce dark current caused by these so-called "hot pixels" the CCD can be cooled. Sensors used in scientific-quality cameras are cooled by forced-air, thermoelectric (Peltier coolers), or by cryogenic means. Every six degrees centigrade reduction in temperature reduces dark current by a factor of two. Dark noise within a CCD is produced by electrons thermally generated within the structure of the CCD, with the largest contribution resulting from the interface between the silicon dioxide and the epitaxial silicon layer in the CCD. Full-frame CCDs operated in multiphase pinned mode can reduce this effect. In multiphase pinned mode, the signal charge is directed away from the interface toward the buried n-channel to reduce dark noise, increase charge-transfer efficiency, and increase pixel-to-pixel uniformity. Hole-accumulation diode photosites used in interline-transfer CCDs have the same effect as multiphase pinned mode clocking. (Roper Scientific CoolSNAP is an example.)

Data Transfer Plug Type:

Transfer plug type is related to the transfer mode and transfer (readout) speed. See Transfer Mode and Readout Speed.

Deconvolution, yes/no:

Camera is useful for deconvolution applilcations. For more information see FAQ

Drivers (VayTek):

VayTek now has native drivers for the QImaging Retiga and the Cooke SensiCam cameras in VayTek's VolumeScan application. VolumeScan provides users with the power and flexibility to control many microscope peripherals, while still having the fastest readout times possible.

Drivers (Image Pro and IP Lab):

Software drivers to control the image acquisition process.

With Image-Pro Plus for PC the user can:

Acquire images easily from video, CCD cameras, scanners, photo CD, scientific instruments and image databases.
Comprehensive file format and device support include CCD cameras, image capture cards, and TWAIN scanner devices.
Image-Pro provides full support of 8-, 12-, and 16-bit precision gray scale, 24-bit color and 32-bit floating point images.
Advanced virtual memory management techniques allow the user to view multiple images, even those which are larger than the display.
Equalization, background subtraction, correction and filed flattening methods enhance color, brightness, and contrast.
Filtering functions sharpen, soften, blur or enhance edges.
Touching or overlapping objects can be separated and morphometric processing can be performed.
Simple geometric measurements and complex structure-function relationships can be obtained.
Absoluted spatial calibration guarantees accurate measurement data.
To complete your research, Image-Pro provides the means to analyze, archive, communicate, and present your images.
For advanced Image processing or OEM product development, use the Image-Pro software development kit.

With IP Lab for Macintosh and PC the user can:

Filter and enhance contrast
Geometric registration, rotate, and scale
Fourier and Cosine transforms
Intensity statistics
Pseudocolor
Profile plots
Identify objects by intensity, size, shape
Count, Label and Measure hundreds of objects in seconds
Density and shape analysis
Time-Lapse and 3D sequence animation with QuickTime movie export
8-bit pseudocolor and 24 to 48-bit full color images
3-D Perspective views
Presentation quality plotting
3-D volume visualization
High Dynamic Range Data:
Capture, process, and display 8-, 16-, 32-bit integer, floating point, 16-, 24-, and 48-bit color data
Interactive brightness, contrast and gamma control
High-quality grayscale and full 24-bit color video frame grabbers
High-resolution 12-16 bit, cooled digital CCD cameras
High-resolution flatbed scanners
Microscope focus control, automatic filter wheels, and XY stage motors
Light sources, shutters, VCRs, etc.
Use IPLab's Scripting mechanism to create macro-functions
Assign frequently used Scripts to Macintosh keyboard F-keys for easy access
Add your own C and Pascal code to create custom Extension software modules

To purchase Image-Pro or IPLab, or to inquire about additional features and technical specifications of , Contact VayTek via email, or phone 515-472-2227.

External Toggle:

A pin on the camera that puts out a 5 volt pulse for controlling a shutter, or accepts a pulse to trigger acquisition.

Fluorescence Microscopy, yes/no:

Camera is appropriate for fluorescence microscopy or not.

Frame Grabber:

The board in the computer that acquires the data from the camera and passes it to the computer. There are two types: analog and digital. Analog cameras require analog frame grabbers, usually RSI-70. Digital frame grabbers vary widely. Some use a standard digital signal (RS 422 or IEEE 1394) and others are proprietary. In our experience, the IEEE 1394 ("Firewire" as trademarked by Apple) and proprietary solutions are the easiest to use. Cameras that use third party digital frame grabbers are frequently a hassle.

Gain Control:

Multiplies the signal (and background) by a constant, making dim images brighter. Gain control can be done on the camera (better and faster) or in software (slower).

Integration Time Increment Unit:

The minimum time the camera will integrate, usually 1 ms or less.

Mount Type:

There are two common mount types: C and F. C is by far the most common.

Pixels, Number of:

The number of effective pixels on the chip.

Resolution Table for Zeiss Objectives
The theoretical limit of the resolving capability of light microscopes is highly dependent on the objective. The following table shows the resolving power of Carl Zeiss objectives in the intermediate image for 0.63 x and 1.0 x TV coupling adapters in combination with a 2/3" CCD (8.5 mm x 6.4 mm).

Objective	Magnification	N.A.	Lp/mm TV-Cpk 1.0X	Necessary Camera Resolution	Lp/mm TV-Cpk 0.63X	Necessary Camera Resolution
Plan-Neofluar	1.25	0.04	96	1632 x 1229	152	2548 x 1946
Fluar	2.5	0.12	144	2448 x 1843	229	3893 x 2931
Plan-Neofluar	5	0.15	90	1530 x 1152	143	2431 x 1830
Achroplan	10	0.25	75	1275 x 960	119	2023 x 1523
Fluar	10	0.5	75	2550 x 1920	238	4046 x 3046
Plan-Neofluar	20	0.5	75	1275 x 960	119	2023 x 1523
Plan-Apochromat	20	0.75	113	1921 x 1446	179	3040 x 2291
Plan-Neofluar M.-Imm	25	0.80	96	1632 x 1229	152	2548 x 1946
Plan-Neofluar	40	0.75	56	952 x 717	89	1513 x 1139
Plan-Neofluar	40	1.3	98	1666 x 1254	155	2635 x 1984
Plan-Apochromat	63	1.4	67	1139 x 858	106	1802 x 1357
Epiplan-Neofluar	100	0.90	27	459 x 346	43	731 x 550
Plan-Apochromat	100	1.4	42	714 x 538	67	1139 x 858

Power Input:

Some cameras support 110 and 220. Some cameras require a separate power supply box.

Precapture Delay:

Sets a delay in the camera that is applied before image acquisition. Precapture delay is useful for triggered events and for physiology experiments.

Price:

List price is shown. Contact us to find out if you qualify for a VayTek discount on the camera of your choice.

Read Noise:

Your specimens are related to the noise and bit depth of the camera you choose. If your specimens are dim and you have to integrate a long time, or you have to capture images quickly, then the noise will be greater in the images. The noisier the image, the less accurate the deconvolution. To minimize camera noise under these circumstances, select a camera with the lowest cooling temperature, the largest well capacity, the lowest dark current and the lowest read noise. Signal to noise ratio is an important parameter. Three sources contribute to signal to noise ratio: photon, dark, and readout noise. Photon noise cannot be reduced by camera design. It is the inherent natural variation of incident photon flux. However, dark and readout noise can be reduced by cooling methods and camera design.
Here's how you can convert Signal to Noise from dB (decibels) into ratios and back again.

Convert dB to ratio
Divide the dB number by 20. Use this number as an exponent of 10 to find the value of the ratio to 1.
Example: 60 dB / 20 = 3. 10^3 = 1000. Ratio is 1000:1

Convert ratio to dB
Find the log of the ratio and multiply by 20
Example: 2000:1 2000 = 10^3.301. 3.301 x 20 = 66 dB

Readout Speed (Transfer Speed):

The rate at which the information on the camera chip is transferred to the computer memory. This rate is given in full frames/second or MHz. For example, a 20 MHz camera will read 20 million bits (not pixels) per second. The faster the readout speed, the higher the noise. Cameras that allow binning or ROIs (Regions of Interest) will read out at the same rate but will get more frames/second because of the smaller frame size. Different cameras have different readout speeds. If you are working with fixed specimens and dyes that do not photobleach easily, then you can use a camera with slower readout times. The analog cameras work at 30 frames/second, but have noisy images. The cooled digital cameras' readout speeds vary from 1 Mhz (considered slow) to 20 Mhz, or higher (for IEEE 1394 Firewire). A 1 Mhz camera will usually produce a full frame image (about 1 million pixels) in about 1.5 seconds. On the other end of the spectrum, the 20 Mhz, or faster, cameras can operate at 10 frames/second or better. Note that a faster readout increases the noise in the image. If you bin an image, or take a subregion, then the frame rates can approach 100 frames/second, or better. If you are working with live specimens, doing calcium ratio imaging, or working with specimens that photobleach easily, you will want the greatest sensitivity and the fastest readout times. Frames per second is abbreviated as "fps" in the comparison database.

Resolution:

One question that is often asked by those who want to do deconvolution is "What resolution should I have for my camera?". This depends on your lens magnification, na, the wavelength of light, the camera chip dimensions, and the camera coupler.

It is easy to compute.

We have the formula for calculating the resolving power of a lens:

1) d = (Lambda/2) * NA

where
d = the smallest resolvable distance between two points under the lens
Lambda = the wavelength of light
NA = the numerical aperture of the lens

The resolvable distance at a conjugate image plane where the camera chip is placed can be calculated with

2) D = d * M

where
D = is the resolution at the image plane
d = resolving power of the lens
M = the magnification of the lens

In order to get the maximum resolution in the image on the camera chip it is necessary to capture two pixels worth of data. The number of pixels per mm can be calculated as:

3) R = 2/D

where
R is the maximum camera resolution in pixels/mm

Combining formulae 1, 2 and 3 we get the formulae:

R(x) =( (N * NA) / M) * (X/T)
R(y) =( (N * NA) / M) * (Y/T)

Where
R = the number of pixels required on the camera in the x or y direction
N = 4000/Lambda (in microns)
NA = the numerical aperture of the lens
M = the magnification of the lens
X = the chip size in the x direction in mm
Y = the chip size in the y direction in mm
T is the reduction or magnification factor of the camera coupler

For example, a 100x, 1.4 na lens projection onto a 2/3" (8.5 x 6.4 mm) camera chip, using a .63 camera coupler and red light (Lambda = .6) would require a camera with a resolution of 760 x 570 pixels.

SDK:

Software Developer's Kit. A kit for those who want to write their own drivers.

Separate Controller Required?:

Computers that have a 6-pin 1394 FireWire port provide power for the camera through the cable. If the computer power supply is unable to provide about 5.5 watts for uncooled cameras, or about 11 watts for cooled cameras, then a 1394 power supply or a 1394 FireWire hub is required. Many camera manufacturers offer external power supplies as an option or accessory. A 6-pin to 6-pin Firewire cable is usually included with FireWire-powered cameras, but 4-pin to 6-pin cables are usually available for computers that have a 4-pin port. Computers with 4-pin ports require an external power supply or hub because the 4-pin port will not provide power. Some 6-pin ports also do not supply sufficient power, especially for cooled cameras. Check the power output specifications of your computer to determine if you must order an optional external power supply or hub.

Shutter Type:

There are two types, mechanical and electronic. The mechanical shutter consists of an iris that is opened and closed by a solenoid. Mechanical shutters are required by some chips because of their readout process. They can't be electronically shuttered. Mechanical shutters are slower and eventually wear out. "Electronic shutter" simply indicates that the camera stops accumulating photons after a certain time (the integration time) and then resets the chip.

Shutter Speed:

In many cameras, the shutter exposure time is linked to the camera exposure. The delay between when the shutter opens and closes relative to the read-out of the CCD may be factory adjustable in firmware only.

Size:

The external dimensions of the camera head.

Spectral Response:

CCD chips have a characteristic response curve depending on the color of the light. For example, the Sony interline chips are more sensitive in the blue-green region of the spectrum, and less sensitive in the red spectrum. If you are doing calcium imaging, then you will probably favor a camera that uses the Sony chip. The Kodak chip, however, is more sensitive the red region. This camera is especially good for dyes like Cy3 and Cy5. You can check the spectral response curve of several cameras by consulting the spread sheet on our web site. You should consider this issue carefully. If you have to integrate longer, then the specimen may bleach over time. This is especially true if you are attempting to acquire a stack of images for 3D reconstruction. The spectral sensitivity of CCD sensors (quantum efficiency) is the percentage of photons converted to electronic signal. The higher the quantum efficiency, the more sensitive the CCD sensor. The spectral response is different for each CCD at every wavelength.

Subregions:

The ability to tell the camera to return a subsection of the chip. Choosing a subregion reduces readout time and the use of disk space. Subregion selection is essential for some applications, including calcium imaging and deconvolution.

Transfer Cord & Connector:

Some cameras require several cables, others just one. This is an ease-of-use issue. You may also want to check to see if the manufacturer includes all the necessary cables in the camera purchase package.

Transfer Mode:

Refers to the process by which the information in the chip pixels are read off the chip and consolidated for readout to the computer. For analog cameras this is not very important, since the signal is converted to analog. For digital cameras, Transfer Mode has important implications. There are three basic modes, two of which can be found in the cameras in our comparison database: full frame and interline. The full frame mode collects the information in each pixel and passes it along to its neighbor until it reaches the final pixel at the end of the row. These values are then read out of the camera from the last row. This readout scheme means that the full chip must be readout before the next image can be acquired. This is a slower method than the interline transfer. The Kodak chip listed in our comparison database uses full frame transfer. The Sony chips use interline transfer. In interline transfer mode every other column is reserved for acquisition and its immediate neighbor is reserved for readout. This means that the camera can be acquiring an image while it is still reading out the previous image. The Sony Chip is, therefore, faster. Unfortunately, the interline transfer method distorts the way the light falls on the chip, a problem which can be compensated for by using a micro lens array to concentrate the light onto the appropriate column.

Transfer Speed (Readout Speed):

Twain Driver:

A universal type of acquisition protocol that can be used with many third party programs, such as Adobe Photoshop. Twain Drivers are good for simple acquisition, but useless for complex applications.

Updatable, Programmable EPROM:

An updatable EPROM means the camera can be loaded with new software to fix bugs and to add new features more easily. This is an important support and upgrade issue.

User Settable Delay:

This feature lets the user set a delay in the camera that occurs before an image is acquired. A settable delay is useful for physiological experiments where a trigger is needed.

VayTek Rating:

Based on our experience with cameras in the real world, we have rated reach camera from 1 (worst) to 10 (best). While these ratings are totally subjective, they are based on our active participation in helping users with various applications over the past twelve years.

What is the best camera for digital deconvolution?

The two most important issues you must consider to answer this question are the characteristics of your specimens and the software you will use to integrate your system. Click on the link above to read VayTek's detailed examination of these issues.

Weight:

Weight is an important factor for some microscope stands. Added wight can cause the stand to flex, bringing the lens closer to the specimen.

Well Depth:

See "Read Noise". To really appreciate the meaning of well depth you have to look at the ratio of the well depth to the read noise. For example, if a camera has a well depth of 40,000 and read noise of 10, it would have a signal to noise ratio of 4000:1. This would mean it would be a true 12-bit camera, i.e. capable of reading out 12 bits of dynamic range (4095 shades of gray) accurately. If a camera has a well depth of 20,000 and a read noise of 10, then the S/N ratio would be 2000:1, or a 10 bit camera (2000 accurate shades of gray).

The tricky part is this: a camera can produce a 12-bit image on your computer, but it may not have an accurate, representative 12 bits of data in the image. You can snap an image from the camera to your computer, then look at the image with your imaging software. It will tell you it is a 12-bit image. However, if the camera has a S/N ratio of 2000:1, technically you have a 10-bit image.

To illustrate, let's compare the Retiga camera and the Spot RT camera. The Retiga camera has a well depth of 16,000 electrons and a read noise of 8 bits. This means it has a S/N of 2000:1. It is a 10-bit camera in terms of the accuracy of its signal to noise ratio, but it will produce a 12-bit image on your screen.

The Spot RT has a rating of 60 db. This is only a little confusing. You can translate db to S/N ratio and do the comparison. Db is defined as 20 log10 R, where R is the S/N ratio. If you do the numbers, you will see that the Spot RT has a S/N of 40,000:40, or 1000:1. This means it is a 9-bit camera, or that it has an effective dynamic range of 1000 shades of gray, even though it will produce a 12-bit image on the screen.

VayTek Inc., 505 North Third Street, Tetra II Bldg, Suite 200, Fairfield, IA 52556
Tel 641-472-2227 Fax 641-472-8131 Email vaytek@vaytek.com

© 2000 VayTek Inc. Information may be reproduced only with permission from VayTek Inc.