Fluorescence microscopes are excellent tools to generate contrast in images of biological samples. However, they suffer from a major problem when you are imaging samples even slightly thicker than the z resolution limit. The problem is the out-of-focus blur. This happens because the system is unable to capture light from precisely the focal plane of the objective. Some out of focus light (light from planes below and above the focal plane) inadvertently creeps in and introduces noise. This results in blurred images.
There are few ways to overcome this problem and get sharper, in-focus images. This helps the microscope to perform precise optical sectioning. The most widely used and probably the gold-standard in optical sectioning is confocal microscopy.
How does the confocal microscope reduce out-of-focus-blur?
A very basic understanding can be had from the description below.
Let us take a laser that emits at the excitation wavelength of the fluorophore of interest. The laser beam is reflected by a dichroic mirror towards an objective. See Figure 29. The beam is a made to fill the back aperture of the objective. This creates a focused, diffraction-limited spot of the excitation light at the focal point of the objective. If there are fluorophores in this focal volume, they will get excited and will emit fluorescence isotropically. A cone of this emitted light (that travels in the backward direction) is collected by the objective. You can see that we are using the epi-illuminaiton configuration here. The objective sends this fluorescence emission light out in a collimated beam shaft (remember Figure 2). Here the focused laser spot creates something similar to a point source of light at the focal point of the objective. This means the beam comes out collimated and is directed towards the dichroic mirror.
The dichroic mirror is designed to reflect the excitation wavelength (from the laser) and transmit the emission wavelength (from the fluorophores in the sample). The dichroic mirror hence transmits this collimated beam shaft of emitted light. A tube lens further down focuses this light to its focal point. If we now place here an emission filter and a photodetector we can measure the intensity of emitted light from the focal spot of the objective. Note that the objective-tubelens combination is acting like an infinite tube length microscope and is projecting a magnified image of the focal spot of the objective onto the detector plane.
The detector measures the intensity of the light emitted from the focal spot. However, we have out of focus light which also gets detected by the detector.
The solution to this problem is a pinhole. The pinhole should be kept exactly at the focal point of the tube lens. This is very important. The detector is placed after the pinhole. The detector used in most common implementations of a confocal microscope is a Photo Multiplier Tube (PMT). This is shown in Figure 30.
Let us now consider what happens to the light coming from the focal spot (Figure 30). If the light comes from a point source placed at the focal spot (refer Figure 2) it comes out collimated through the back aperture of the objective. Now a collimated beam of light enters the tube lens. The tube lens focuses the light to its focal point. The pinhole is placed exactly here. Almost all of the emission light that originates from the focal spot passes through the pinhole and is detected by the PMT.
Now let us consider the out of focus light. First, the light that comes from a point at a distance shorter than the focal length of the objective. Look at Figure 8 from left to right. If the light comes from a point source placed at a distance shorter than the focal length, the light exits the lens slightly diverging. This scenario for a confocal microscope is shown in Figure 31.
Note that when we use high NA objectives the out of focus light it is not produced over large distances away from the focal point but probably a couple of micrometers above and below the focal point. This is the range of distances over which the objective collects fluorescence light. Beyond these distances, the intensity of excitation is very low to excite any detectable fluorescence. Hence the divergence of the beam as the light exits the lens is not very large.
This divergence, however, is significant enough to have an influence on how the beam exits the tube lens as is seen in Figure 31. Lets us consider Figure 6. Look at this figure from right to left. For that matter, you will see the same thing in Figure 4 and Figure 5 when looking at them from right to left. (Remember lenses are reciprocal systems, a ray diagram is true in both directions).
When a diverging beam enters a lens, it gets focused at a distance that is longer than the focal length of the lens. Now this means the light is not focused all the way down when it hits the pinhole (Figure 31). The pinhole hence blocks the light coming from a point on the sample that is at a distance shorter than the focal length of the objective.
Lets us now consider the situation when the objective collects light from a point that is at a distance longer than the focal length of the objective. Look at Figure 6 again but this time look at it from left to right. You will see the same thing in Figure 4 and Figure 5 as well when looking at them from left to right. If the light comes from a point source placed at a distance longer than the focal length of the objective, the light exits the lens converging. Again, in a confocal microscope, this light is collected only from very short distances away from the focal point. So the light out of the objective is only slightly converging. This scenario is shown in Figure 32.
Now a converging beam is passing through the tube lens. Lets us consider Figure 8. Look at this figure from right to left. When a converging beam enters a lens, it gets focused at a point that is at a distance shorter than the focal length of the lens. Now this means the converging light in the confocal microscope is focused before the pinhole by the tube lens. After being focused as light propagates further it diverges and the pinhole blocks this light as well.
So the combination of the objective, tube lens, and the pinhole blocks most of the out of focus light allowing only in-focus light to be detected by the PMT.
We have however collected intensity information from only a single diffraction limited spot. This does not create an image. To create an image, we need to scan the laser beam in a raster pattern and collect intensity information from many points across the XY plane. This creates a 2D array of intensity information stored in the memory of the computer. This array is then converted into an image.
How is the point scanning confocal microscope system configured?
The block diagram of one of most common implementations of a point scanning confocal microscope is shown in Figure 33. The diagram gives a more detailed description of the components involved in making a confocal microscope work.
The excitation light for a point scanning confocal microscope comes from a laser combiner. The laser combiner has a bank of lasers with different wavelengths. The different wavelengths are required to excite fluorophores with different excitation wavelengths. These wavelengths are combined into a single collinear beam using a set of mirrors and dichroic mirrors. This combined laser beam is passed through an Acousto Optic Tunable Filter (AOTF). AOTF is controlled electronically through the software that runs the confocal microscope. The AOTF serves two functions:
- Select a wavelength or a set of wavelengths for excitation
- Control the intensity of each wavelength independently
The light out of the AOTF is launched into a single mode optical fiber. This arrangement helps the laser combiner to be separated from the scan head and imparts flexibility in placing the laser combiner with respect to the scan head. The other end of the optical fiber is connected to the scan head. The excitation light diverges out as it exits the optical fiber. A collimating lens is used to collimate this light. The collimated output is directed towards the dichroic mirror.
The dichroic mirror reflects the excitation light towards the scanning mirrors. The scanning mirrors scan the excitation wavelength/s in X and Y dimensions in a raster pattern. The scanning mirrors are controlled electronically through the sofware of the confocal microscope.
Before directing towards the objective, the light excitation light out of the scanning mirrors is passed through a set of two lenses called the scan lens and the tube lens. The tube lens sits in the body of the microscope and the scan lens inside the scan head. These two lenses have the following functions:
- Together they act like a telescope to expand the laser beam from about few mm to about 1 cm. This is to ensure that the back aperture of the objective is overfilled. This, in turn, ensures that the full Numerical Aperture of the objective is available and maximum possible resolution is attained.
- Relay the image of the scanning mirrors to the back aperture of the objective. This ensures a proper scanning of the excitation laser spot across the defined ROI on the sample plane.
As described before the objective focuses the excitation light onto the sample. The fluorescence emission (in the backward direction) is collected by the objective. The fluorescence emission traces back the path taken by the excitation light all the way till the dichroic mirror. The dichroic mirror transmits the emission light. A tube lens (of the scan head) focuses the emission light onto the pin hole. The light out of the pinhole is filtered by an emission filter and directed towards the PMT.
The PMT produces a current that is proportional to the number of emission photons detected by it. A Trans Impedance Amplifier (TIA) is used to convert this current into a voltage and to amplify the voltage to appropriate levels. An Analog to Digital Converter (ADC) is used to convert the analog voltage signals into digital signals. Computers can only read digital signals. The intensity information in digital format is read by the computer and it converts this information into an image.
How does a confocal microscope generate an image?
Widefield fluorescence microscopes use imaging detectors like CCD or sCOMOS cameras for generating images. Cameras have an array of detectors in them which corresponds to the pixels we see in the images they generate. If a camera is specified as 5 Mega Pixel, then the camera chip has 5x10^6 independent detectors. When the lens system of the widefiled microscope projects an image onto the detector plane, such a camera can generate a digital image with 5x10^6 pixels.
However confocal microscopes use single pixel detectors like PMTs. Single pixel means they donot have an array of detectors but only a single detector. So how are these detectors generate images?
The fact is in a confocal microscope the image is not generated by the detector. It is generated by a computer that has a certain amount of memory and processing power. The image is constructed by the computer using a set of intensity information sequentially gathered from the PMT and sequentially stored in the memory of the computer.
The laser spot is scanned in the raster pattern and spot moves across the XY plane. As the laser spot steps from one spot to next, the output of the PMT changes every set time period. This time period is the Dwell Time-the time it takes the scanning system to move the laser spot from one pixel to the next on the sample plane. The ADC is programmed to take a reading of the PMT voltage (through TIA) every time period. So every successive time period (dwell time) the ADC spits out a digital value. This value corresponds to the intensity of fluorescence emission from the position of the laser spot at that particular time point.
Before you start imaging you need to tell the confocal software how many pixels you need in your image. If you say you need 512x512 pixels, the computer allocates a memory location in the form of an array of size 512x512. Each memory location has an address ([0,0] to [511,511]). Let us assume that an ROI is scanned. The scanning system positions the laser on the first spot (let's say on the top left of the ROI). The ADC reads the PMT output and generates a digital value. This corresponds to the intensity of fluorescence from the first spot. The computer stores this value in the array at memory location [0,0]. The laser spot is then moved right to the next spot. The ADC reads the value and this is stored in memory location [0,1], then the next one onto [0,2] and so on till memory location [0,511] is filled. This completes scanning of the first row. Now the laser spot is moved to the next row and the process repeats. Starting from [1,0] the memory locations are progressively filled till [1,511].
So as the laser beam progressively moves across the sample plane the memory locations get progressively filled up. This happens until the laser spot reaches the last point in the ROI on its bottom right. This fills the last cell in the array with address [511,511] and created a completely filled memory array. This array of intensity information is called a bit map. This bitmap is converted into an image in a given format like JPEG, PNG, TIF etc. or custom formats proprietary to confocal microscope manufacturers. This image can be displayed on a computer monitor or stored.
This whole process requires precise synchronization between the scanning system and the ADC. This is done by the control electronics which works under the command of the confocal software. Modern confocal microscopes donot step the laser spot across the XY plane. The laser spot instead is moved continuously in a raster pattern. The ADC simply reads periodically (every dwell time) from the PMT.
How does a confocal microscope generate 3D images?
To generate a 3D image, a 3D array is defined with the number of pixels in X, Y and Z directions specified. After the image of one plane is scanned, the relative position between the objective and the sample is changed by the Z step size. This is done by moving the objective or moving the sample placed on a Z stage. Z step size is calculated from the number of pixels defined in the Z direction and thickness of sample needed to be imaged. This process is repeated for multiple planes and a raster scan performed and an image generated for each plane. The number of image planes equals the number of pixels in the Z direction. The end result is a 3D array of intensity information. This array can be volume rendered or projected onto a single plane for visualization.