At the heart of digital imaging is the image sensor. This consists of an array of light-sensitive receptors, embedded into a microchip containing the wiring and circuitry necessary to record light levels captured from each receptor. The receptors, termed pixels (an abbreviation of picture-elements) generally consist of photodiodes embedded in a well. The photodiodes convert photons of light striking the sensor into electrons in a proportional relationship (the more photons striking, the more electrons generated). The charge generated is measured by the microchip circuitry, converted to a digital signal and processed by in-camera software.
There are a number of different sensor types. The first generally available is the Charge Coupled Device (CCD). The CCD is still the most common type of sensor in professional photomicrographic cameras but has largely been replaced in consumer cameras by the Complimentary Metal Oxide Sensor (CMOS). The CMOS sensor is present in a greater range of consumer devices; mass-production means that the CMOS sensor is significantly cheaper than the equivalent CCD sensor. Although the CMOS sensor requires more processing by the camera software to reduce noise in the image, the image quality from current generation of CMOS sensors cannot be distinguished from images created by CCD sensors.
Resolution and Image Quality
Camera manufacturers can vary both the number of pixels on a sensor, and their individual (physical) size; more pixels can be accommodated into a sensor chip of given size by reducing the dimensions of each pixel. Resolution – the ability to see two adjacent points in the image as separate – is determined by both the number of pixels and their size. For the two adjacent points in the specimen to be recorded as individual elements in the image, the microscope must first of all be able to resolve the points. The objective must be of sufficiently high numerical aperture to resolve the structure, and the microscope must be correctly set up; the highest resolution digital camera cannot record information that is not present in the optical image. Secondly, the two adjacent points in the image must fall onto separate pixels, and these pixels must be separated from each other to show a ‘gap’ between the two structures
Larger pixels have the advantage that they can capture more light before becoming saturated and have a higher signal to noise ratio; they are thus more appropriate for recording images of low light intensity where long exposures are necessary. Smaller pixels capture less light before becoming saturated and have a higher signal to noise ratio, but provide greater image resolution. In most situations, much of this is academic; the choice of camera will be based on cost, and sensor resolution in terms of the number of pixels.
Quality of the image depends on the output device – computer screen, projector, or print. Understanding the relationship between image resolution and output device is perhaps the most misunderstood aspect of digital photography with the terms ‘PPI and DPI’ used interchangeably. PPI stands for ‘Pixels Per Inch’ and determines the size of the image on the output device. The number of pixels used to record the digital image file is fixed by the camera sensor; this cannot be changed. However, these can be displayed or printed at a variable number of pixels (each represented as one coloured dot in the image) per inch. Lower PPI values will result in a larger output image size but the ‘dot’ nature of the image will become more apparent as the PPI value is lowered and the final image will have a ‘grainy’ appearance. For professional printing (such as a book or magazine) 300 ppi is the required quality; printing of photographs on a domestic printer will generally be at 180 to 200 PPI and will be of acceptable quality; graininess will start to become apparent below this value. Computer monitors (PC) generally work up to 96 PPI. Table 1 summarises maximum output image size for various uses of the image.
Choice of Digital Camera
Often digital photomicrography is attempted with a pre-existing camera and almost any digital camera can be utilised to record images from the microscope; several ingenious arrangements to couple less-suitable cameras to microscopes have been published. However, if selecting a new camera there are several important considerations.
The most fundamental decisions relate to how the camera is to be used, and how the images are to be used. If the camera is to be used for general photography as well as photomicrography, a conventional consumer level compact camera or digital single lens reflex (DSLR) is necessary to provide a lens function. Alternatively if the camera can be wholly dedicated to the microscope, a specialised microscope camera system may be appropriate. However, it is the balance between sensor resolution (number of pixels) and cost that is probably most important. Digital cameras have evolved rapidly in their short existence; a ten-fold increase in sensor resolution of consumer level cameras has been achieved in as many years whilst prices have dropped in real-terms. Almost any modern digital camera will now provide more than adequate resolution for all but magazine quality publishing.
The next question relates to overall sensor size. The physical dimensions of the sensor will have an impact on how much of the field of view of the microscope can be projected onto the sensor and therefore recorded. Larger sensors will record more of the visual field of view, assuming no change in projection optics. The situation is however, complicated by compact cameras that have a built-in zoom lens and several images with smaller fields of view can now be ‘stitched’ together by software to make a larger overall image. Figure 1 shows the variation in physical sensor size from a 1.3 MP webcam to a 21.1 MP full-frame sensor; Figure 2 shows the impact on field of view of each sensor.
| Both sensor resolution and sensor size have a major impact on cost of the camera; the third (and often over-riding question) in selecting a camera is ‘how much do I want to spend?’
The cheapest starting-point for digital photomicrography is a dedicated webcam (Figure 3). These are supplied with optics to replace the microscope eyepiece and simply drop into place in the microscope tube. Connection to a computer via a USB link provides power to the camera and software included with the camera provides the control. Both still and video images can be recorded to the computer. The great advantage of webcams is their low cost and ease of use. Against this, sensor resolution is very low and the camera can only be used in combination with a computer. A typical webcam of 1.3 MP will produce prints of tolerable quality up to 6” x 4”.
A wide range of compact digital camera is now available and some are more suitable for photomicrography than others. In looking for a model suitable for use with a microscope, the following features are important:
- A female screw thread to the front of the lens designed to allow filters or lens adapters to be fitted. This thread can be used via an adapter, to connect the camera to the microscope.
- Zooming of the lens is achieved through internal movement and the front lens element does not protrude in front of the lens barrel beyond the filter screw thread.
- Availability of a remote control to trigger the shutter without physical contact with the shutter-button (which would cause vibration to occur)
- Remote control of the camera functions directly from a computer via a USB link
For photomicrography, compact digital cameras have many advantages; low cost, high sensor resolution (especially in ‘top end’ cameras that match the resolution of digital SLRs), and relative ease of use. The main disadvantage comes from the fixed camera lens which is less flexible than a removable-lens DSLR and can introduce optical artefacts (such as ‘hot spots’ or ghosting) in the recorded image. These can often be eliminated in many instances by trial and error if a range of projection optics are available; there seems to be no way of determining if a particular camera and projection lens is compatible other than trying it. Undoubtedly the best approach is to follow a recommendation on model of compact digital camera from an existing microscope user, either through personal contact or via an internet forum.
Arguably the best ‘all-round’ camera for photomicrography is the DSLR. The removable lens allows for easy coupling using a T-mount adapter and photomicrographic systems designed for use with 35mm film cameras (and now available at very low cost second-hand) can be used, providing advantages such as a focussing telescopes (still more accurate than using the camera or computer screen) and vibration-free shutter mechanisms. Most currently available DSLRs have CMOS sensors with resolution of at least 12 MP; sensor size is usually smaller than 35mm film and is often based on the APS-C film format of approximately 22 x 15 mm (the exact dimensions vary between manufacturers). The main disadvantage of DSLRs is the vibration that can occur from movement of the camera mirror when the shutter is released. For low magnifications, or with long (> 3 seconds) exposures this has little practical impact but for shorter exposures or higher magnifications, the vibration can cause blurring of the recorded image.
In selecting a DSLR for use with the microscope, the following features are desirable:
- Live View. This function is available on a growing number of DSLRs available on the market and allows a real-time image to be displayed on the camera screen or, if coupled to a computer with appropriate software, on the computer screen. Live View makes focussing much more reliable than viewing through the camera eyepiece; most DSLRs do not have interchangeable focus screens designed for finding accurate focus at high magnifications. The ‘refresh’ rate of Live View is much lower than for video and therefore it is less useful for tracking and focussing rapidly-moving objects under the microscope; in these instances coupling the camera to a relay lens system with focussing telescope is to be preferred.
- Remote control of the camera from a computer via a USB link. Some manufacturers include this software free with the camera but others do not and the software can be an expensive addition. There are also excellent ‘third party’ remote control software programmes (see ‘Image Capture’ below) but it is wise to check compatibility before buying a camera.
- Some cameras (e.g. Canon) have a ‘silent’ Live View option (usually buried in the ‘Special Settings’ menus). In conventional Live View mode, the camera mirror flips into the ‘up’ position and the mechanical shutter opens allowing the image to be ‘seen’ by the digital sensor and displayed. When the shutter button is triggered, mechanical shutter closes and then re-opens to provide the correct exposure; in some systems the mirror also flips down to allow exposure sensors located in the reflex housing pentaprism to determine the correct exposure. These actions induce some vibration which can caUse slight blurring of high magnification images. Designed to prevent camera shutter noise from scaring wildlife, silent Live View mode utilises an electronic shutter that does not require the mechanical shutter to first close (or the mirror to flip down) to make an exposure. For the microscopist, silent Live View mode is desirable for higher magnification photomicrography. More information and results of trials on Silent Live View can be found on Charles Krebs’ website, listed under Resources).
- The ability to record video. Several DSLRs released in the last year now have the ability to digitally record high quality video. This can be of great use to the microscopist working with live specimens or dynamic processes such as crystallisation.
Dedicated digital cameras are made by the major microscope manufacturers (as well as dedicated specialist imaging companies). These systems tend to be of lower resolution and higher prices than consumer level cameras but are of very high sensitivity to cope with the low levels of fluorescence and confocal microscopy. Such systems (by virtue of their price) are generally not used outside of specialist laboratories but are slowly becoming available on the second-hand market. Potential buyers should be very cautious of the risks involved with these electronic systems, sold outside of the manufacturer’s support or warranty. Lower cost dedicated microscope cameras are available via internet auction websites but reports of image quality from some systems have been disappointing.
Table 2 summarises the relative merits of each class of digital camera.
Connecting Camera to Microscope
The simplest solution to bring camera and microscope together is the dedicated microscope webcam. These directly replace the microscope eyepiece and drop into the microscope tube; adapters are usually provided for wider diameter stereomicroscope tubes.
DSLR cameras have a removable lens and usually require a T2-mount adaptor which connects to the camera lens bayonet and provides a universal female M42 thread. This can then be coupled to a conventional microscope camera adapter with relay or projection optics (Figure 5).
Many microscope adapters (e.g. the Pentax adapter) consist of a simple tube without relay optics; it is inappropriate to use an eyepiece designed for visual use with these adapters as these are designed to project an image at infinity (and brought to a focus by the lens of the eye). To project a real image onto the camera sensor, refocusing will be necessary which introduces spherical aberration, and degrades the image. The correct solution with these simple tube adapters is to use a specially designed projection eyepiece; however good results can often be obtained by simply raising the visual eyepiece by a few millimetres using a cardboard collar).
In many ways, the ideal solution for coupling a DSLR to the microscope is to use a second-hand photomicrographic system; these can often be bought on internet auction sites at very low cost.
| Fig 4
Replacing the film body with the DSLR provides the benefit of much greater precision of focus (using the eyepiece rather than the camera or computer screen) and can effectively eliminate the vibration problem with DSLR cameras. This is achieved by first triggering the DSLR shutter, with a shutter speed of several seconds, waiting two or thee seconds for vibration from the mirror to subside, then triggering the photomicrographic system shutter, timed for correct exposure.
The lens on compact digital cameras however, cannot be removed and some ingenuity is required to connect these to a microscope. With fixed lens camera, it is appropriate to use a visual eyepiece on the microscope; the camera lens performs the same function as the eye’s lens in focusing the image onto the photosensitive receptor (the eye’s retina or the camera sensor). The eyepoint of the eyepiece should be at least 15mm above the upper surface of the eyepiece in order to avoid vignetting in the image. Often these ‘high eyepoint’ eyepieces are designed for use by spectacle-wearers and are marked with an icon of a pair of glasses. The most widely adopted solution is to use a microscope eyepiece that has a screw thread, allowing eyepiece and camera to be directly connected (in most instances) via a screw thread adaptor. The Leitz Periplan x10/20 GF eyepiece (marked with model number 519 815) serve this purpose well, having a plastic eyecap which is screwed to the top of the eyepiece. Removal of this reveals a 28mm male screwthread which can mate directly to a female lens filter mount on the camera of the same thread, or to an interconnecting ‘stepping’ adapter. Other models of Periplan eyepiece are also suitable, but may have a different screw thread and the upper surface of the lens projects above the screw thread, requiring a short spacing tube between the eyepiece and camera lens to prevent the two optical surfaces coming into contact. Periplan eyepieces are no longer available new but can be obtained from internet auction sites (although prices are rising dramatically). Adapter rings can also be purchased from internet auction sites or from specialist companies such as SRB-Griturn. Other suitable eyepiece systems are also available secondhand and it can often be a matter of trial and error to establish a workable system. Care should be taken with any system to evaluate images carefully for flare, ghosting or hotspots in the final picture.
Software for Digital Photomicrography
The digital photomicrographer need not have any software (or indeed a computer) to record digital images; files can be printed out directly from camera storage media on many domestic printers or at high street processors. However, to achieve the full benefits and potential that the digital image offers, a range of software tools is essential
The most basic software requirement is the driver file for the camera. This enables the camera and computer to communicate when linked by cable (or wireless transmission) and enables basic functions such as image download. This software is supplied with the camera from new and is often available as a free download from the manufacturer’s website. Although it can be possible to download images without the relevant camera driver, other camera functions will not be controllable from the computer without it. Those buying secondhand digital cameras should ensure that the driver software is included, or at least downloadable from the internet.
The most beneficial software is the image editing programme. It is not the intent of this article to review the many packages that are available, varying in cost from free shareware to advanced and complex systems costing several hundred pounds; general books and magazines on photography should be consulted to review the market. However, the author uses Adobe’s Photoshop Elements and, like many other users, has found this package to be relatively simple to use, reliable and more than adequate in functionality. The image editing programme provides a wide range of functions; those important to the photomicrographer include image cropping, exposure management, colour management, cloning to remove dust marks and sharpening. Many editing programmes also include photo album organisers – electronic management of digital images into album-like folder structures. Unless many thousand photographs are to be stored, the author has found these to be unnecessary as the required level of management and backup can be achieved with a simple folder structure, rigorously maintained. A further advantage of image processing software is the ‘Panorama-Merge’ function. Designed to blend a number of adjoining pictures of (typically) a landscape into a single panorama, this can be used to great benefit in photomicrography where the field of view is insufficient to record the whole specimen, or where it is desirable to use a higher numerical aperture objective to achieve high resolution. Systematic imaging of the specimen according to a grid pattern (using the X and Y controls of a mechanical stage), ensuring overlaps of each image, allows large specimens to be recorded at high resolution.
Many digital cameras can be controlled directly from a computer via a USB link and this offers many benefits to the photomicrographer; preview of the image on the computer screen, control of basic camera functions such as ISO setting, colour temperature setting, metering mode, exposure control, remote triggering of the camera shutter and near-instantaneous download of the image for immediate viewing. Some manufacturers (e.g. Canon) include this software ‘bundled’ with the camera; others provide this as an accessory, often at high additional cost. Third party ‘remote capture’ software is also available for purchase via the internet, of which the best known system is Breeze Systems’ DSLR Remote Pro software. In the author’s experience third party software can provide much better functionality and interface than the camera manufacturer’s own software.
| Fig 7
Low cost image stacking software is one of the most significant advances in recent years, enabling stunning images with high depth of field to be easily produced; very useful for cladocera. Professional, and expensive, systems that process multiple images have been available for some years; the functionality of these systems is now available in free or low cost software. Systems currently available include Combine-Z, Helicon Focus and Zerene-Stacker. The author uses Helicon Focus which provides a very easy to use interface, with effective results. Other Zerene-Stacker as providing images greater flexibility in use. A workflow for using image stacking software is provided in Figure 7.
Taking the Picture
Digital cameras are no substitute for good microscopy! The first step to a good photomicrograph is to set up the microscope correctly with even illumination (Kohler illumination for compound microscopes) with no glare. Assuming the camera has been correctly mounted with an eyepiece as described above, focus should be achieved either on the camera screen (Live View mode) or computer screen if shooting tethered to a PC. In more advanced adapters a viewing eyepiece is present and should be used to focus. The camera should be set to a low ISO speed (e.g. 100 ASA) and set to either manual exposure control or shutter-priority. If mirror lockup is available (DSLRs) this should be enabled in the camera menu and a test shot taken. Results can quickly be seen on the camera screen or computer (tethered shooting) and examined. Common initial defects are poor focus (readjust), uneven illumination (adjust lighting or condenser) or incorrect exposure (try again).
Like drawing, practice makes perfect but persevere; failure with digital cameras at least costs nothing to try again
Philip M. Greaves
This article is adapted from a fuller version first published in the Quekett Journal of Microscopy, 2010, Vol. 41 Part 4.
Digital Photo-Microscopy by D. J. Jackson. Volume III of Better Microscopy A Series of Practical User’s Guides, 2008. Obtainable from www.lulu.com.
General guidance and support:
www.quekett.org.uk (website and forum of the Quekett Microscopical Club)
SRB-Griturn Ltd. Unit 21D, Icknield Way Farm, Tring Road, Dunstable, Beds LU6 2JX, Telephone 01582 661878.
Remote Capture Software:
Image Stacking Software: