Magnification is the ability of a microscope to produce an image of an object at a scale larger (or even smaller) than its actual size. Magnification serves a useful purpose only when it is possible to see more details of an object in the image than when observing the object with the unaided eye. At the present time, magnification is well defined when viewing an image of a sample through the eyepieces of a microscope. For this case, rigorous international standards have been documented [5–9]. Many of these standards also apply to digital microscopy, but strict definitions and standards for magnification achieved by a digital microscope, where the image is most often viewed by display on an electronic monitor, are still in development.
Digital microscopes, as well as microscopes for visual observation equipped with digital cameras, allow the rapid acquisition of high quality images. Often they are used for a variety of technical applications [10, 11], in many different fields and industries.
Magnification in digital microscopy
What exactly is magnification? A basic definition of magnification is the ratio of the size of a specific feature of an object or sample as seen in an image produced by an optical system to the actual size of the feature on the object itself. Thus, lateral magnification, MDIS, can be defined as:
It should be noted that the useful range of perceived visual magnification significantly depends on the maximum resolving power of the microscope system. When the magnification passes beyond the useful range, then no additional details about the sample can be seen. This situation is referred to as empty magnification [12, 13]. Based on the maximum resolving power, also a useful range of viewing distance, i.e. the distance between the digital display and the observer’s eyes, can be defined for practical reasons.
Digital microscopes or microscopes for visual observation with digital camera
When observing the image through the eyepieces of a microscope for visual observation, the total (lateral) magnification is defined as :
- MTOT VIS is the total lateral magnification observed through the eyepiece,
- MO is the objective lens magnification,
- q is the total tube factor (zoom and other tube lenses), and
- ME = eyepiece lens magnification.
For the case of detecting an image of a microscope which is projected onto an electronic sensor, such as that of a digital camera, the magnification for the image formed at the sensor is :
- MTOT PROJ is the (lateral) magnification of the microscope (image projected onto sensor),
- p is the projection factor from eyepiece to camera, and
- MPHOT is the magnification of the photographic projection lens from the tube to camera.
The total tube factor, q, is normally between 0.5 : 1 and 25 : 1. The photographic projection lens magnification, MPHOT, is normally between 0.32 : 1 and 1.6 : 1.
For digital microscopes, there are no eyepieces, so an image is projected onto and detected by an electronic sensor of a digital camera, and then displayed onto an electronic monitor for observation. This fact is also true for a microscope for visual observation equipped with a digital camera when the image is observed via the monitor. Thus, the final total magnification for digital microscopy, MDIS (Equation 1), will always depend on the actual pixel size of the monitor. It can be defined as:
where MDIS is the total lateral display magnification for an image displayed on a monitor and pixel ratio is the "enlargement" of the image due to the signal transmission of the image from the camera to the electronic monitor display.
The pixel size ratio is determined by the ratio of the pixel size of the monitor to that of the camera sensor:
Here a display of the image from the camera chip to the monitor is assumed to occur in a 1-to-1 pixel correspondence mode, the simplest case scenario. In this display mode, depending on the monitor’s number of pixels, only a portion of the image may visible on the monitor.
Examples of digital microscopy are shown in Figure 1: a digital microscope and stereo microscope with digital camera.
Fig. 1: a) Leica M205 C stereo microscope equipped with Leica DFC450 C digital camera. The ant sample can be observed via the eyepieces or an electronic display monitor for image detection by the camera. b + c) Leica DMS1000 digital microscope utilizing different monitor sizes for image display.
For optical instruments in general, resolution is the ability to see fine details in an image. More specifically, resolving power is the ability to distinguish in an image adjacent points or lines of the object which are closely spaced together. Usually these two terms are used synonymously, however resolution is the more practical one. In microscopy, resolution is expressed in line pairs per millimeter. In other words, pairs of black and white lines with equal line thickness and spacing can be distinguished at a given resolution.
High magnification values without sufficient resolution lead to empty magnification, as already mentioned above [12, 13]. Therefore, it is of vital importance to understand the limiting factors for resolution, not just for digital microscopy, but all forms of optical microscopy.
Camera sensor and display monitor pixel number and size
The sensors used in all microscope digital cameras produced by Leica Microsystems have a number of pixels typically between 1,600 × 1,200 and 4,080 × 3,072 and a pixel size between 2 and 6.5 μm (examples in Table 1). High definition (HD) computer monitors or televisions have 1,920 × 1,200 or 1,080 pixels and a pixel size between 0.1 and 0.9 mm (examples in Table 2) [14, 15]. Therefore, the monitor pixels are typically 25 to 450 times bigger than the camera pixels.
|Camera||Sensor type||Width (mm)||Height (mm)||Pixel size (µm)||Pixels||Megapixels|
|DFC450||2/3''||8.7||6.5||3.4||2,560 × 1,920||4.92|
|MC120 HD/DMS300||1/2.3''||6.1||4.6||3.34||1,824 × 1,368||2.5|
|MC170 HD/DMS1000||1/2.3''||6.1||4.6||2.35||2,592 × 1,944||5.04|
Table 1: Specifications of image sensors used in the Leica DFC450 and MC120/170 HD digital cameras and the Leica DMS300/1000 digital microscopes.
|HD flat display||Width (mm)||Height (mm)||Pixel size (mm)||Pixels||Megapixels|
|PC monitor 21.5''||476||267||0.25||1,920 × 1,080||2.07|
|PC monitor 24''||521||324||0.27||1,920 × 1,200||2.3|
|PC monitor 27''||597||337||0.31|
1,920 × 1,080
3,840 × 2,160
Table 2: Examples of HD electronic monitor displays: computer monitors or TVs.
Pixel size ratio
From knowing the typical pixel sizes of the camera sensors (Table 1) and flat screen HD monitors (Table 2), then values for the size ratios can be easily calculated using Equation 5 (Table 3).
|Camera type||Monitor size (inch)|
|DFC450||144 : 1||132 : 1||253 : 1||218 : 1||162 : 1||106 : 1||91 : 1||79 : 1||74 : 1|
|MC120 HD/DMS300||147 : 1||135 : 1||258 : 1||222 : 1||165 : 1||108 : 1||93 : 1||81 : 1||75 : 1|
|MC170 HD/DMS1000||209 : 1||192 : 1||366 : 1||315 : 1||234 : 1||153 : 1||132 : 1||115 : 1||106 : 1|
Table 3: Pixel size ratios (Equation 5) for HD monitors (Table 2) and the Leica DMS1000/DMS300 digital microscopes and Leica MC170/MC120 HD and DFC450 digital cameras (Table 1).
Examples: Digital microscope and stereo microscope with digital camera
For simplicity, only 2 examples of digital microscopy, actually a digital microscope and a stereo microscope equipped with a digital camera, will be discussed in this report. It is assumed that an image is displayed, using a 1-to-1 camera to monitor pixel correspondence, onto a HD monitor with sizes ranging from 21.5'' (diagonal dimension 21.5 inches [54.6 cm]) to 75'' (diagonal dimension 74.5 inches [189 cm]). The 2 examples are the digital microscope Leica DMS1000 and the stereo microscope Leica M205 A having the digital camera Leica MC170 HD installed with a C-mount. Table 4 shows examples of total magnification (refer to Equations 2 and 4) values obtainable with the Leica DMS1000 or M205 A equipped with the MC170 HD camera. For the Leica DMS1000, the magnification range for the objective lens is 0.32x to 2x and the tube factor (q) including the photographic projection lens has a 8.4 : 1 range. For the Leica M205 A with MC170 HD camera, the magnification range for the objective is 0.5x to 2x, for the zoom 0.78x to 16x, for the eyepieces 10x to 25x, and for the C-mount lens 0.4x to 1x.
|Leica DMS1000||Leica M205 A/MC170 HD|
|Monitor size (inch)||Eyepiece||Monitor size (inch)|
|8.4 : 1||29 : 1||min.||3.9||9.75||16.5 : 1||57 : 1||min.|
|420 : 1||1,450 : 1||max||320x||800x||3,400 : 1||11,700 : 1||max.|
Table 4: Total magnification data, MTOT VIS and MDIS (Equations 2 and 4), for the Leica DMS1000 digital microscope and the Leica M205 A stereo microscope equipped with the Leica MC170 HD digital camera. The possible range of magnification values, minimum to maximum, for the discussed HD monitor sizes (Table 2) and pixel ratios (Table 3).
30,000 : 1 magnification
Which monitor pixel size would be needed to attain a total lateral display magnification of 30,000 : 1? An example can be shown using the Leica M205 A with MC170 HD camera and Equations 3b, 4, and 5. The maximum magnification for the M205 A for an image of the sample projected onto the camera sensor is:
The pixel ratio value which corresponds to a total magnification of 30,000 : 1 with the above magnification of 32x onto the sensor is:
The pixel size of the Leica MC170 HD camera sensor is 2.35 μm. Using the pixel ratio value above, 938 : 1, and a 1-to-1 camera to monitor pixel correspondence, the monitor pixel size must be:
Therefore, to achieve a total magnification of 30,000 : 1 with the Leica M205 A and MC170 HD digital camera, the monitor pixel size would have to be 2.2 mm. This pixel size would correspond to a HD monitor diagonal of 4.9 m!
Useful range of magnification for digital microscopy
Now one must ask the question if this level of magnification, 30,000 : 1, is simply beyond the useful range, meaning it is empty magnification. How do we determine a useful range of magnification for digital microscopy, where an image is observed from a monitor? First it is important to understand better the microscope system resolution and the viewing distance.
Microscope system resolution
The system resolution for a digital microscope (or stereo with digital camera) is influenced by 3 main factors:
- Optical resolution:
where NA is the numerical aperture and λ is the wavelength of light in nm;
- Image sensor (camera sensor) resolution:
where MTOT PROJ is the magnification from the sample to the sensor (Equation 3), the "sensor bin. mode" refers to the binning mode which is 1 for full frame, 2 for 2 × 2 pixel binning, etc. (refer to Figure 2), and "pixel size" refers to the sensor pixel size in µm; and
- Image display (monitor) resolution:
where MDIS is the total lateral magnification (Equation 4) and the monitor pixel size is in mm.
The basis for the camera sensor and display monitor resolution limit is the Nyquist rate or frequency from the sampling theorem for digital signal processing (refer to Figure 2) . This theorem assumes that at least 2 pixels are needed to resolve 1 line pair. For this report, as stated above, the best case scenario of a 1-to-1 correspondence is assumed between the pixels of the sensor and monitor. Therefore, using Equation 4 and converting the monitor pixel size into units of µm, it becomes clear that the resolution limit of the sensor and monitor are identical.
The resolution limit of the digital microscope system resolution is determined by the smallest of the 3 resolution values above.
Figure 2a–b: a) Examples of pixel binning modes for camera sensors when detecting images: no binning (full frame, 1 × 1), double binning (2 × 2), triple binning (3 × 3), and quadruple binning (4 × 4), and b) Camera sensor detection of black/white line pairs, used to measure the resolution limit of a microscope, requires a minimum of 2 pixels (red squares) per line pair (Nyquist rate). However, better image results are obtained if 3 or more pixels per line pair are used.
Useful range for viewing distance
The viewing distance is the distance between the observer’s eyes and the displayed image. The useful range for the viewing distance is affected by the system resolution of the microscope and visual resolution angle of the observer . The latter is normally 2.3 to 4.6 minutes of arc for typical human eyes. In other words, a human eye is capable of distinguishing details on a monitor which have a separation distance corresponding to an angular difference of more than 2.3 to 4.6 minutes of arc for a specific viewing distance. The useful range for the viewing distance can be expressed as:
where MDIS is the total lateral magnification (Equation 4) and "system resolution" refers to the microscope resolution limit as discussed above.
For the discussion here, it is assumed that the viewing distance is always within the useful range.
Useful range of magnification
To understand how to determine the useful range of magnification for digital microscopy, i.e. the observation of a magnified image on a display monitor, it is first necessary to mention briefly the perceived magnification from visual observation of an image or object. Using geometrical optics, the following can be derived:
where MDIS is the total magnification (Equation 1) and 250 refers to the standard reference for the viewing distance in mm which is based on the average near point for the human eye.
Now, finally the useful range of magnification can be defined by combining Equations 9 and 10:
Thus, the useful range of magnification is between 1/6 and 1/3 of the microscope system resolution.
Modern camera sensors have pixels sizes in the 1–6 µm range, well below 10 µm. When a high sample-to-sensor magnification is used, for example 150 : 1, and there is no binning of the pixels and a 1-to-1 sensor to monitor pixel correspondence, then it follows from Equations 6, 7, and 8 above that the microscope system resolution is determined by the optical resolution limit. The optical resolution limit for the largest numerical aperture, approaching 1.3, and the smallest wavelength of visible light, approximately 400 nm, is 5,400 line pairs/mm. For these same conditions, the resolution limit of a camera sensor with a pixel size below 10 µm easily exceeds this value. For the conditions of this specific case, from Equation 11 above the maximum magnification in the useful range of values is 1,800x.
At low magnification from the sample to camera sensor, 1x or even less, numerical apertures are typically below 0.03. The resolution limit of camera sensors with pixels sizes larger than 2 µm will start to be inferior to the optical resolution at such low magnification. Therefore, at low magnification, 1x or less, the sensor or monitor resolution limit will likely be the dominating factor concerning the resolution of the microscope system.
For this discussion of digital microscopy, it is assumed that the image on the monitor is always observed within the useful viewing distance range described above. Whenever the perceived magnification value exceeds the useful magnification range, i.e. 1,800x, then no further details about the sample can be resolved.
Object field (field of view)
Object field (OF) is the part of the object which is reproduced in the final image. It is also known as the microscope field of view (FOV). Thus, details of an object can only be observed if they are present in the object field.
When looking through the eyepieces, the OF is a visible circular image of a portion of the sample. The size of the OF (refer to Equation 12) is dependent on the field number (FN) of the eyepiece, as well as, the magnification of the objective and tube lenses (refer to Figure 3).
The object field in digital microscopy is of rectangular shape due to the nature of the image sensor which receives the image and the monitor which displays it (refer to Figure 3). It is expressed in width and height given in millimeters. For digital microscopy, care has to be taken that the image created by the optical system is large enough to cover the whole image sensor. In this case, the OF can be limited either by the image sensor or the display. In either case the physical size of the active area, given by the number of active pixels in height and width and their physical size (pixel pitch), has to be taken into account.
To calculate the OF, the physical size of the active area of the sensor (refer to Equation 13) has to be divided by the magnification of the objective, tube, and camera projection lenses (MTOT PROJ) or for the monitor by the total lateral display magnification, MDIS. The smaller of these values for each height and width define the OF of the digital microscope.
It is likely that both height and width of the object field are not necessarily jointly limited by the image sensor or the display. For example, the height can be limited by the display whereas the width can be limited by the sensor. The final OF will depend on the dimensions and aspect ratio of the image sensor and display and the pixel correspondence (1 : 1, 1 : 2, 2 : 1, etc.) between them for image display. In this report, a 1-to-1 sensor pixel to monitor pixel correspondence is assumed.
The OF for eyepieces can be determined by:
- OFeyepiece is the object field observed through an eyepiece,
- FN is the eyepiece field number, and
- MO × q (from Equation 2) is the total magnification before the eyepiece due to the objective, zoom, and any other tube lens before the eyepieces.
The OF for a camera sensor can be determined using the width and height of the sensor divided by the total magnification of the optics producing the image of the sample onto the sensor:
- w is the width of the OF observed by a sensor,
- h is the height of the OF observed by a sensor,
- MTOT PROJ is the total magnification from sample to sensor (Equation 3b), and
- the pixel size is in μm.
The difference in OF between images seen by the eyepieces versus those recorded by the camera chip, for the same sample, objective, and zoom setting, are shown in Figures 3 and 4 below. For Figure 4, the total magnification of the objective and zoom lens is 1x, but several types of Leica C-mounts with different magnification have been used to install the camera, a Leica DFC450 with a chip size of 2/3'' onto a Leica M205 A stereo microscope. The red rectangle seen in Figure 4a represents the OF of Figure 4b, an image taken with the 0.32x C-mount. The blue rectangle indicates the OF of Figure 4c, taken with the 0.5x C-mount. The green rectangle shows the OF of Figure 4d, taken with the 0.63x C-mount. Figure 4b shows the problem of vignetting where the edges of the image are darker than the center. To avoid such a problem, normally it is recommended that a 0.32x C-mount is used with a digital camera having a 1/3'' (8.45 mm) chip size, a 0.4x C-mount with a 1/2.3'' (11 mm) chip size, a 0.5x C-mount with a 1/2'' (12.7 mm) chip size, and a 0.63x C-mount with a 2/3'' (16.9 mm) chip size.
Figure 3: Diagram showing direct comparison of an image viewed through the eyepieces (white circle) and simultaneously with the chip (rectangles) of a digital camera from Leica Microsystems. The 2 examples shown are: a) eyepiece with a field number (FN) of 20 mm and C-mount with 0.4x lens and b) eyepiece with 23 mm FN and C-mount with 0.5x lens. Some cameras detect images in a 4 : 3 aspect ratio (red rectangle) format for data storage and a 16 : 9 aspect ratio (green rectangle) format for live image output.
Figure 4: Images of a Siemens star taken with a Leica M205 A stereo microscope having a total objective and zoom lens magnification (MO × q) of 1x. The 1st black line circle has a 10 mm diameter and the 2nd a 20 mm diameter (seen in 4a and 4b). Images are a) photographed through a 10x eyepiece with 23 mm FN and recorded using a Leica DFC450 digital camera equipped with a Leica C-mount (MPHOT): b) 0.32x MPHOT and 27.2 × 20.3 mm OF; c) 0.5x MPHOT and 17.4 × 13 mm OF; and d) 0.63x MPHOT and 13.8 × 10.3 mm OF. The red rectangle in 4a represents the OF of 4b (0.32x C-mount), the blue the OF of 4c (0.5x C-mount), and the green the OF of 4d (0.63x C-mount).
The object field (OF) of the camera sensor can be calculated using Equation 13 above. The range of values of the OF for the Leica DMS1000 and Leica M205 A equipped with a MD170 HD camera are shown in Table 5. Again, the magnification range for the Leica DMS1000 is: objective 0.32x to 2x and the tube factor (q) including the photographic projection lens has a 8.4 : 1 ratio, and for the Leica M205 A with MC170 HD camera: objective 0.5x to 2x, zoom 0.78x to 16x, and C-mount 0.4x to 1x.
|Leica DMS1000||OF sensor|
|MTOT PROJ||w (mm)||h (mm)|
|0.08 : 1||77||58.1||max. OF|
|3.96 : 1||1.5||1.2||min. OF|
|Leica M205 A/MC170 HD||OF sensor|
|MTOT PROJ||w (mm)||h (mm)|
|0.16 : 1||39.1||29.5||max. OF|
|32 : 1||0.19||0.14||min. OF|
Table 5: Object field (OF) data (Equation 13) for an image from the Leica DMS1000 digital microscope and Leica M205 A stereo microscope equipped with a MC170 HD digital camera showing the range from minimum to maximum values.
Summary and conclusions
Digital microscopes use electronic image sensors (camera sensors) to replace eyepieces. Microscopes for visual perception, such as stereo microscopes, have eyepieces and can be equipped with digital cameras. Digital microscopy allows rapid acquisition of high quality images. It is often used for fast and easy documentation, quality control (QC), failure analysis, and research and development (R&D) in a variety of fields.
Due to the diversity of camera sensor dimensions and electronic display monitor sizes, determining magnification and resolution when using digital microscopy can be challenging. With this report users of digital microscopy can better understand how to evaluate the total magnification and its useful range. In addition, helpful information concerning the object field or field of view is discussed.
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