HOW LFM WORKS
From light to a 3D view.
Follow the light through brightfield, fluorescence and confocal microscopy. See how a scan becomes an image, and a stack becomes a view through depth.
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Full transcript
Back to the film ↑The complete spoken narration, arranged by chapter.
01 How light becomes a picture
How can light reveal something too small for our eyes, and even help us look through a cell? Let's follow the light. We'll use original, synthetic specimens, not recordings of living cells. Start with transmitted light, then switch to fluorescent labels, and finally build a three-dimensional view from a stack of images.
02 Brightfield: light passes through
In brightfield, a condenser directs light through the specimen. Structures that absorb more light appear darker against the bright background. Transparent, unstained cells can be difficult to see this way. The objective collects light from many specimen points. In this microscope, another lens, the tube lens, brings the corresponding image points onto the camera. The camera's many pixels measure arriving light during an exposure. Close the shutter. The image disappears, but the lamp can stay on. The shutter blocks the path; it does not switch off the source.
03 Bigger is not always sharper
Now move focus slowly. The selected plane becomes sharp, then blurred. Focus selects a depth; magnification makes the view larger. Enlarging a blurred picture cannot restore missing detail. Compare these two close points at the same displayed size. With a wider accepted cone of light, their blurred images become narrower and easier to distinguish. That light-gathering cone is described by numerical aperture, or N A. Higher N A can preserve finer detail under comparable conditions. Wavelength, illumination, the specimen and image sampling also matter.
04 Light the field, not just the centre
Two illumination controls have different jobs. The field diaphragm limits the area receiving light. Closing it does not shrink the cells. The condenser aperture changes the illumination cone. More contrast is not automatically more detail. Keep the field evenly illuminated before comparing brightness.
05 Fluorescence: labels make selected structures visible
Now we change the preparation and contrast mechanism. These synthetic fluorescent cells are a different teaching specimen from our brightfield example. Fluorescent labels absorb excitation light and emit light at longer wavelengths. Here, the green channel represents a label associated with actin. Actin does not naturally glow green. Follow the blue excitation route through the objective. Returning fluorescence travels back through the same objective. A colour-selective mirror, called a dichroic, and an emission filter separate the returning signal from excitation light. Choose the wrong emission filter: the image becomes dark even though the specimen is still illuminated.
06 Widefield or confocal?
In widefield fluorescence, many positions are illuminated together, and a camera records the image. Light from nearby depths can blur together. Keep the same fluorescent specimen for the next comparison. A point-scanning confocal microscope focuses laser excitation into a small spot and uses a detection pinhole. It reduces light coming from other depths, making the selected plane easier to inspect.
07 A scan and a pinhole build one section
Scanning mirrors move the focused spot across the specimen: along a row, then onto the next. Fluorescence returns through the objective and scanning mirrors toward a stationary pinhole. Light from the focused spot is concentrated near that opening. Out-of-focus light spreads more widely there, so much of it is blocked. Some still passes. The pinhole acts on returning light; closing it does not dim the excitation reaching the specimen. A smaller opening rejects more background, but also loses useful photons. After the pinhole, a point detector measures brightness. It does not take a whole photograph. The computer combines each brightness measurement with the scanner's position to build the image. The unvisited pixels have not been measured yet. These paths are unfolded for clarity, and the scan is slowed to make it visible.
08 Sections become a sampled 3D volume
That image is one optical section. Nothing has physically cut the cell. Its depth sensitivity is soft, and neighbouring sections overlap. Now move focus, scan another section, and save its depth. Repeat to create a Z stack. Every measured point has a position across the image, a depth, and an intensity. Together, these samples support a three-dimensional intensity view of the selected label. The reconstruction cannot recover structures the microscope did not record. Smaller Z steps sample depth more densely; they do not automatically make the optical sections thinner. A projection can place structures from different depths on top of one another. Inspect individual sections too. More planes also take more time and, at unchanged settings, more light dose.
09 Your turn in MVLscope
Try these discoveries in How LFM works. Change one control and predict what will happen. Compare focus, try a filter, build a scan, then explore a stack. In real fluorescence microscopy, more illumination can bleach labels and damage living specimens. Aim for useful information, and pause illumination when it is not needed.