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how do you increase depth of field on a microscope

how do you increase depth of field on a microscope

It depends on type of the microscope – optical/SEM/TEM/STM – and the specific hardware.If the focus range is long enough increase the working distance to get bigger depth of focus.If you have a condenser aperture in your system go to a smaller aperture size.You want to decrease your collection angle – meaning get your beam more parallel.You can use focus stacking. It is a way to combine similar pictures at lower DOF to create a picture of larger DOF. This video details the procedure.The depth of field is a function of the relationship between the image magnification and the diaphragm opening (aperture).You will have to reduce the aperture at that magnification or reduce the magnification at that aperture


.Changing the lens focal length to affect d.o.f. from a given subject-camera distance is changing the image magnification, in effect.To make a field stop, cut/or punch a clean circular hole in an opaque (black is better) stiff paper or thin card. You can use a sharpie, or india ink to make an index card black enough, too. Maybe you can find the right-sized metal or fibre washer in a hardware store or misc. parts you may already have around your studio, or lab, shop or house – paint it black to cut down flare. Tape it in place over the lens.Place your aperture in front of the microscope lens centering the aperture. For some gross-specimen low-magnification ‘scopes, this is relatively easy since the lens is quite large.The field-stop acts as an outboard aperture to limit the light entering the lens to the centre.


The effect is increased apparent depth due to the “stopping down” (reducing the aperture) of the lens.Experiment to find the right aperture to achieve the depth of field you wish. The exposure will have to compensate for the reduction of light. There might be some vignetting, but c’est la vie.
The background could be a bit lighter, too, which might help the image detail rendition.


What is the depth of field in microscopy?


In various scientific fields, a microscope is an important tool in viewing and examining objects that are too small to be seen by the naked eye. One of the basic components of a microscope is an eyepiece or ocular lens. A nosepiece, which has several objective lenses is located below the eyepiece. The objective lenses are calibrated into different levels of magnification. Depending on what magnification the specimen is to be viewed, the nosepiece can be easily rotated and the objective lens set into place. Other parts include a mechanical stage, substage condenser, iris diaphragm, substage illuminator, a rheostat and two knobs that are adjusted to focus the sample specimen.

When viewing an object, the overall magnification is calculated by multiplying the value of the ocular lenses with the value of the objective lenses. The total magnification is indirectly proportional to the depth of field. As magnification increases, the axial resolving power of an objective, which is the depth of field, is reduced.

Depth of Field and Depth of Focus


When considering resolution in optical microscopy, a majority of the emphasis is placed on point-to-point lateral resolution in the plane perpendicular to the optical axis . Another important aspect to resolution is the axial (or longitudinal) resolving power of an objective, which is measured parallel to the optical axis and is most often referred to as depth of field.

Axial resolution, like horizontal resolution, is determined only by the numerical aperture of the objective , with the eyepiece merely magnifying the details resolved and projected into the intermediate image plane. Just as in classical photography, depth of field is determined by the distance from the nearest object plane in focus to that of the farthest plane also simultaneously in focus. In microscopy depth of field is very short and usually measured in units of microns. The term depth of focus, which refers to image space, is often used interchangeably with depth of field, which refers to object space.This interchange of nomenclature can lead to confusion, especially when the terms are both used specifically to denote depth of field in microscope objectives. The geometric image plane might be expected to represent an infinitely thin section of the specimen, but even in the absence of aberrations, each image point is spread into a diffraction figure that extends above and below this plane. The Airy disk, a basic unit of the diffraction pattern produced by the microscope objective, represents a section through the center of the intermediate image plane. This increases the effective in-focus depth of the Z-axis Airy disk intensity profile that passes through slightly different specimen planes.

Depth of focus varies with numerical aperture and magnification of the objective, and under some conditions, high numerical aperture systems (usually with higher magnification power) have deeper focus depths than do those systems of low numerical aperture, even though the depth of field is less (see Table 1). This is particularly important in photomicrography because the film emulsion or digital camera sensor must be exposed or illuminated in a plane that falls within the focus region. Small errors made to focus at high magnification are not as critical as those made with very low magnification objectives. Table 1 presents calculated variations in the depth of field and image depth in the intermediate image plane in a series of objectives with increasing numerical aperture and magnification.

At high numerical apertures of the microscope, depth of field is determined primarily by wave optics, while at lower numerical apertures, the geometrical optical circle of confusion dominates the phenomenon. Using a variety of different criteria for determining when the image becomes unacceptably sharp, several authors have proposed different formulas to describe the depth of field in a microscope. The total depth of field is given by the sum of the wave and geometrical optical depths of fields as:

Where d(tot) represents the depth of field, λ is the wavelength of illuminating light, n is the refractive index of the medium (usually air (1.000) or immersion oil (1.515)) between the coverslip and the objective front lens element, and NA equals the objective numerical aperture. The variable e is the smallest distance that can be resolved by a detector that is placed in the image plane of the microscope objective, whose lateral magnification is M. Using this equation, depth of field (d(tot)) and wavelength (λ) must be expressed in similar units. For example, if d(tot) is to be calculated in micrometers, λ must also be formulated in micrometers (700 nanometer red light is entered into the equation as 0.7 micrometers). Notice that the diffraction-limited depth of field (the first term in the equation) shrinks inversely with the square of the numerical aperture, while the lateral limit of resolution is reduced in a manner that is inversely proportional to the first power of the numerical aperture. Thus, the axial resolution and thickness of optical sections that can be attained are affected by the system numerical aperture much more so than is the lateral resolution of the microscope.

The human eye can normally accommodate from infinity to about 25 centimeters, so that the depth of field can considerably greater than that given by the equation above when one observes the microscope image through the eyepieces. On the other hand, a video sensor or photographic emulsion lies in a thin fixed plane so that the depth of field and axial resolution using those sensors are given by the parameters in the equation. In these cases, the axial resolution is defined by convention as one-quarter of the distance between the first minima, above and below focus, along the axis of the three-dimensional diffraction image produced by the objective.
Figure 2 – Depth of Field versus Numerical Aperture,These values for the depth of field, and the distribution of intensities in the three-dimensional diffraction pattern, are calculated for incoherently illuminated (or emitting) point sources where the numerical aperture of the condenser is greater than or equal to that of the objective. In general, the depth of field increases, up to a factor of 2, as the coherence of illumination increases (as the condenser numerical aperture approaches zero). However, the three-dimensional point spread function (PSF) with partially coherent illumination can depart in complex ways from that so far discussed when the aperture function is not uniform.

In a number of phase-based, contrast-generating modes of microscopy, the depth of field may turn out to be unexpectedly shallower than that predicted from the equation above and may yield extremely thin optical sections. In digital and video microscopy, the shallow focal plane in the target of the camera tube or CCD, the high contrast achievable at high objective and condenser numerical apertures, and the high magnification of the image displayed on the monitor all contribute to reducing the depth of field. Thus, with video, we can obtain very sharp and thin optical sections, and can define the focal level of a thin specimen with very high precision.



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