OPTICAL MICROSCOPES are widely used in the life sciences as
well as the physical sciences. This note is principally directed
at life science work and is a general guide to applications for
life scientists wishing to improve the quality of the imaging
work they undertake with optical microscopes.
Optical microscopy may use transmitted light, including brightfield,
phase contrast and darkfield microscopy (where the light detected
is passed from a light source through the specimen onto the detector);
it may use fluorescent emission (where a short wavelength light
source is used to illuminate the specimen and the fluorescent
emission from the specimen is detected); or it may use luminescence
(where the specimen is self-luminous).
In addition to conventional microscopes, the confocal microscope is increasingly used. These have the advantage of being able to suppress light (transmitted in some cases but mostly fluorescent) that is seen as a diffuse background from out-of-focus parts of the specimen in order to give sharper images. Confocal microscopes can give beautiful images but they do require rather high light levels, levels that may be reduced substantially with the use of PixCellent CCD imaging systems.
In many cases the needs of the optical microscopist will be well met from the Standard range of CCD cameras sold by PixCellent , but some specialist applications will need systems selected from the Specialist Products Listings.
THE MAJORITY of microscopes are used with the naked eye. This is suitable where a specimen simply has to be checked for the presence or absence of some relatively obvious feature. Film (Polaroid or 35mm) is often used to keep a more permanent record. Television cameras are often used for greater convenience although they offer much poorer image resolution and image quality than the human eye. TV cameras are also now used with frame grabbers attached to a computer system. These boards grab and digitise a single TV frame and pass it to the host computer for display and, increasingly, analysis. There is no doubt that these systems allow many useful tasks to be undertaken routinely but it is also true that the quality of the results obtained often leave much to be desired. The image resolution is usually poor, with 256 x 256 or 512 x 512 pixels being typical, whereas the eye can see about 2000 x 2000 pixels. In addition, the eye is able to see very low contrast features that these frame grabbers often miss. This is because the grabbers generally digitise the data to 8-bits or 256 grey levels. In practice, if the light level is carefully set so that the signal peak is close to the grabber peak level then it is possible to see features which differ in brightness by 1 to 2 % from their surroundings. However the eye is capable of much better performance. It has automatic exposure control and the ability to filter out substantial background variations and detect low contrast features that most TV systems simply fail to detect. PixCellent specialises in camera systems for microscopy that give a performance that greatly exceeds the performance of the human eye. They use solid-state detectors called charge-coupled devices (CCDs) that are available with very high resolutions and with exceptional low contrast capabilities (from 1:4000 to 1:1,000,000).
The data generated are completely linear so for the first time accurate, repeatable quantitative measurements are routinely available allowing much more precise work to be undertaken with much less reliance on purely qualitative assessments of the microscope images.
WITH A PixCellent imaging system the microscope is set up in the normal fashion. The camera is fitted to the port where a conventional film camera would be fitted - virtually all standard research microscopes have at least one place to mount a camera, possibly needing a simple adaptor available from your microscope manufacturer. As with an ordinary film camera the image of the specimen is magnified by the microscope objective directly onto the focal plane of the PixCellent camera. In turn the camera is connected to a computer of the IBM/PC Pentium class via a small box of control electronics. The computer runs PixCel, PixCellent 's easy-to-use software package running under Windows™ 95, to capture images and display them for the user, to store images for later use and to perform various types of analysis on the data
THE CAMERAS MADE by PixCellent differ principally in their read-out speed. The Capella system allows very fast read out at several million pixels (picture elements) per second. It has 4000 grey levels (optionally 16000 grey levels) per pixel and can take many high-resolution images per second. The Antares system gives images of the very highest quality. It allows significantly lower contrast features to be detected because it takes images with 65,000 - 1,000,000 grey levels per pixel. It achieves this by reading the camera more slowly at 20 to 167 kHz giving full resolution images in a few seconds. Both cameras are able to read out a small specific area of the detector to give a much faster frame rate, so, for small areas, the Antares is capable of several frames per second.
The next factor is the choice of detector itself, the CCD. CCDs with large numbers of pixels are significantly more expensive, but even the lowest cost CCDs that PixCellent offers (such as the Kodak 0400) already have many more elements than there are in any TV picture. In fact, the Kodak 0400 has 768 x 512 pixels and will give superb images for many applications. It is also the case that CCDs with more pixels take longer to read out, are more demanding on computer memory, disk storage and processing time, so think carefully before specifying too large a CCD. On the other hand, even very large CCDs can easily be run at low resolution by 'binning' neighbouring pixels together and this significantly reduces the read-out time. You may then use the highest resolution only when you need to.
The CCDs that PixCellent offers come with pixel sizes from 6.8 microns to 26 microns square. The larger pixel devices allow higher magnification objectives to be used that have better light gathering efficiency. They also have a larger signal capacity in each pixel so if either high signal levels are to be used or particularly low contrast features are to be studied you might want to consider devices with the larger pixel sizes. Generally both larger numbers of pixels and larger pixel sizes will increase the overall system price. However, larger pixels take no longer to read out than small ones. The Capella fast read-out camera can only be used with the Kodak range of CCDs and the range of EEV frame transfer CCDs although there is a wide variety of device sizes to choose from within the range. PixCellent 's cameras normally operate with the CCD cooled. Unless you are working with chemi-luminescence or bio-luminescence (see below) you should only need to consider thermo-electric cooling and ignore the possibility of liquid nitrogen cooling. In many instances with fluorescent microscopy t is possible to obtain excellent images with uncooled heads. PixCellent include a series of uncooled heads within their standard range for this sort of application.
The thermo-electric heads have either a low vibration, fan assisted heat sink for heat dissipation or one that uses recirculating liquid that can be at room temperature or below as required. The choice between air and water cooling does not affect the system price (although a water circulator, if needed, is extra).
FROM THE POINT of view of PixCellent 's product range, optical microscopy is a relatively high light level application unless you are working in luminescence which is one of the lowest light level applications we deal with. Below are listed some of the main aspects of each sort of microscopy that you should bear in mind while selecting the best imaging system for your application. Do not forget that most of our systems work extremely well without modification in most of these applications. Also do not think that you will get trapped by a decision you will make now. All our systems are highly modular. It is possible to upgrade your system easily and economically to provide more resolution or speed as you require. However, do try to think where your research programme is likely to go in the longer term. You will be impressed at the quality and beauty of the images a PixCellent camera will give you and will soon want to exploit that quality efficiently and effectively. We at PixCellent are committed to helping you with whatever new ideas you might want to try out, and our feature rich software package is likely to have most of the tools you will ever need.
In this mode the specimen is viewed with light transmitted through it. Transmission microscopy covers brightfield, darkfield and phase-contrast microscopy as well as some less common variations. Colour filters may be used to optimise the visibility of the features of interest. The important reason for choosing a PixCellent imaging system for this application is that they have much greater spatial resolution than TV systems. PixCellent cameras also have a much better sensitivity to very low contrast features that are very difficult or impossible to see with a standard TV camera. It is sometimes difficult to stain certain structures on microscope slides and this gives very low contrast features. PixCellent cameras are particularly suitable for detecting and quantifying such low contrast features because of their extreme grey scale resolution (4000 to 1,000,000 levels per pixel).
Here the specimen is labelled or marked with a fluorophore. The fluorophore is stimulated by flooding the specimen with short wave illumination (usually in the ultra-violet, though many fluorophores are excited at wavelengths in the blue, green or red part of the spectrum). The sample then emits light at a longer wavelength which may be filtered to suppress unwanted light at the excitation wavelength. The Antares series of systems offer the highest sensitivity of low contract features and the widest dynamic range.
Many microscopists have severe operational problems in practice.
The fluorescence may be weak and viewed against considerable non-specific
fluorescence from the sample, the microscope slide, the cover
slip glass and sometimes even from the optics of the microscope
itself. For this reason the microscopist is often forced to increase
the UV illumination level so much that the fluorophore is affected
and eventually damaged by over-excitation. This process is called
photo-bleaching. It is irreversible, and greatly affects the quality
of a study in fluorescence microscopy. Hence the experimenter
cannot easily distinguish whether a feature is real or simply
a consequence of partial photo-bleaching. In addition, the levels
of illumination can easily be high enough to damage the specimen,
especially if it is of living tissues or cells. The UV light level
used is often 10-100 times the level from sunlight in the middle
of a desert.
PixCellent imaging systems have a very
great contribution to make here to improve the quality of images
that fluorescent microscopists take. From our point of view, fluorescence
microscopy is a high light level application, and exposures of
only a few seconds are sufficient to give excellent images. PixCellent CCD systems are very sensitive
and have excellent low-contrast capability so microscopists can
use much lower levels of illumination, reducing photo-bleaching
and improving specimen viability. The CCDs have excellent far-red
sensitivity so the microscopist can use the new red fluorophores
that are coming onto the market which work at wavelengths that
minimise non-specific fluorescence. Most of our cameras have much
higher resolution than is possible with TV formats and the wide
dynamic range allows much lower contrast, fainter features to
be detected and accurately measured.
PixCellent is able to offer optional
hardware for minimising photo-bleaching by shuttering the exciting
light source to allow it to illuminate the specimen only when
the camera is making an exposure. In cases where the specimen
is rapidly changing (such as in calcium imaging), the Capella
system allows many full-resolution frames to be taken each second
and even higher frame rates are possible with the programmable
sub-array read-out capability. The Capella systems transfer their
data directly to computer memory across the fast PCI bus. Because
of this they are able to capture data in quantities limited only
by the computer memory capacity.
In these luminescent applications the light detected is emitted
directly by the specimen, either because of the chemicals used
to generate light as part of the energy exchange mechanisms in
the specimen, or directly from living cells that have this firefly-like
capability. In most cases the light levels are extremely low.
PixCellent has considerable experience
in solving many of the problems associated with using enhanced
chemi-luminescent techniques, and can help expert users in this
field directly. The low light levels make the larger pixel CCDs
more efficient, and as long exposure times are normally required,
it is often essential to use either a water-cooled TE head or
a compact liquid nitrogen head for the ultimate sensitivity. There
are two chemi-luminescent methodologies. One gives most of the
light out in a short burst of 5-20 seconds and requires the active
chemicals to be injected onto the specimen and immediately imaged.
The other takes 5 minutes to reach its peak and then the emission
lasts 20-80 minutes. This latter chemistry (used by Amersham in
its Amerlite products) can justify the use of a liquid nitrogen
system whereas a thermoelectric head is optimal for the former
methodologies. The Antares series is the best camera system for
this application as it is the most sensitive and read-out speed
is seldom critical.
The fastest developments in microscopy recently have been in the study of changes in the chemistry of living cells. The original development at the University of Cambridge of the Fura fluorescent probes for the measurement of intracellular calcium is well known, but many other probes allow the measurement of intracellular chloride, sodium and acidity (pH); others allow surface membrane potentials to be measured. In all these cases there is a considerable emphasis on measurements being made several times per second and for these applications the Capella imaging system from PixCellent is especially well suited. Other studies involve the location and identification of marker molecules such as monoclonal antibodies that interact with the immunological sites on the surfaces of cells.
There are a great number of specific examples of these techniques as work. All are directed at discovering how the cell structures and chemistries work in the organism, but the experiments have to be carried out in the hostile environment of the microscope slide, illuminated by an exceedingly intense level of UV radiation.
PixCellent imaging systems can contribute greatly to these studies because they allow the microscopist to reduce the illumination levels dramatically and so improve the life expectancy (viability) of the cells. Even when the cell is not likely to be killed, the ability to limit the stress on a cell or a specimen is critical to obtain unbiased measurements. The dynamics of calcium and other cellular ions is of vital importance to many aspects of cellular function and precise studies are critical to our understanding of these issues. The PixCellent Capella is uniquely suited to these studies because a sequence of images may be taken rapidly at fixed intervals or at intervals that can be programmed exactly to match the dynamics of the phenomena under investigation. The NeuroCam camera from the Standard Range provides many of these facilities.
Confocal microscopes have an important role to play in fluorescent
microscopy studies since they allow very sharp images to be taken.
By using a scanned light source (such as a laser) and only detecting
light from the illuminated spot (and not the surrounding areas)
confocal microscopes are able to suppress significantly the light
from out-of-focus fluorescent objects and from the background.
With appropriate processing they allow 3-dimensional images to
be generated. This can also be done with standard non-confocal
imaging systems by stepping the focus control, taking an image
at each position and again processing to produce a 3-dimensional
image. However, confocal microscope systems are relatively slow
and require intense light levels, usually of green laser light
(Argon, at 488nm). The laser colour limits the fluorophores that
may be used and the extreme light levels needed greatly increase
the risk of cellular stress and death. Because confocal microscopes
are now the latest technology, they are often used for studies
in which a conventional microscope with a PixCellent
camera would give better results at very much reduced cost. Confocal
microscopes are usually fairly slow, with a read-out rate similar
to that of the basic Antares system or slower. They often will
only produce data with 256 grey levels (8 bit digitisation) and
correspondingly poor signal-to-noise ratio images. They are usually
programmable to give single sub-array read-out for speed. The
Capella cameras are able to work well at read-out rates that are
up to 100 times faster than many confocal microscopes. With a
PixCellent camera microscopists work
with a conventional microscope which they may already have and
to which they are likely to be accustomed. A PixCellent
imaging system is an effective and much lower cost system when
attached to a standard fluorescence microscope and will give performance
in many applications that considerably exceeds that from a confocal
microscope. This is especially the case when time resolution is
needed together with good spatial resolution. Most calcium studies
look for 1-10 frames per second for 2-30 seconds. This is difficult
to achieve with confocal microscopes and the gain in spatial contrast
(which is the main advantage of the confocal microscope) may be
lost by the poor time resolution that they give.
PixCellent imaging systems increasingly
have an important place as the detector for confocal microscopes
as they allow the illuminating light level to be minimised. PixCellent cameras also offer 4000-1,000,000
grey levels compared with only 256 levels from confocal microscopes.
Microscopists may have a requirement for a variety of additional features to enhance their systems. Most microscopes can be fitted with motorised stages (x-y motion), motorised focus controls, colour filter wheels and shutters over the illuminating (excitation) light source. These are supplied both by the manufacturers of the microscope and by third parties. PixCel has the built-in capability to allow control of these mechanisms via an RS-232 port in the back of the computer (COM2 for example). It is best if the customer chooses these accessories for himself and if they want PixCellent to handle the complete control, send information to PixCellent about the interface and the level of control needed.
The mechanical shutters that are standard on PixCellent cameras are not able to produce shutters times shorter than 10-20 milliseconds. For a few specialist applications faster speeds are desirable. Also for some applications any vibration can be a problem and the user may wish to avoid using a mechanical shutter. We are now able to offer a liquid crystal shutter for use with our cameras. This is a solid-state shutter that is electrically driven and has no moving parts. It works by polarising the light and then rotating the plane of polarisation inside the shutter one way or the other. This means that the peak transfer efficiency is only 30 % and the output light is polarised (not generally a problem unless a strongly polarised light source is being used and this is seldom the case in microscopy). We operate the liquid crystal shutter (made for PixCellent by Displaytech, USA) as follows: the mechanical shutter is opened with the liquid crystal (LC) shutter closed, the LC shutter is pulsed open for the required exposure time which can be as short as 100 microseconds, and the mechanical shutter is then closed. For fast time resolution, multiple frames may be taken without closing the mechanical shutter between images. This allows much faster frame rates than are achievable with mechanical shutters, and has no vibration problems.
It is very helpful in reducing photo bleaching problems if the illuminating light source is shuttered off until the exposure is to be made and for the light source to be switched on only for the duration of the exposure. PixCellent is able to provide an additional shutter (mechanical, liquid crystal or both) that may be slaved in parallel with the camera shutters and to provide a manual over-ride for set-up purposes. Please contact PixCellent with details of your requirements and your microscope.
PixCellent has considerable experience
of using computers that are networked together. We will be happy
to advise you should you want help in connecting the computer
running PixCel and your PixCellent imaging system
to other machines via a network (e.g. Ethernet).
PixCellent has considerable experience of working with life scientists in general and with microscopists in particular. We often work with researchers whose programmes of research are very much state-of-the-art. PixCellent is keen to help our customers working in these areas and can draw on a very large body of experience. Should you have an application that at first sight looks rather unusual please contact us directly. It is very likely that we will already have come across a similar application and can advise on how it is best handled.
We can also design and manufacture custom hardware to support
a customer's application. Please contact your local distributor
or PixCellent directly with details of
the application and the support hardware or software that may
be needed.