One type of microscopy apparatus is an heterodyne detected optical imaging apparatus, which generates images of a sample based on the principle of vibrational or electronic spectroscopy without contacting or destroying the sample. These types of imaging apparatus are useful for imaging samples including biomass, pharmaceutical samples, lipid bodies, and nanomaterials, among other types of samples. Additionally, heterodyne detected optical imaging apparatus enable imaging of a sample without requiring labeling or staining of the sample.
In use, heterodyne detected optical imaging apparatus generate an electrical output signal that includes an amplitude modulated image data signal and a direct current signal from a local oscillator. Typically, the image data signal is extracted from the electrical output signal by a complex device referred to as a lock-in amplifier.
Lock-in amplifiers, also known as phase-sensitive detectors, have been in use since approximately 1961. Essentially, a lock-in amplifier is a phase-sensitive bandpass amplifier with a variable central frequency and bandwidth. Accordingly, the lock-in amplifier rejects the background signal (i.e. the direct current signal from the local oscillator), filters the image data signal from electrical noise, and then amplifies the image data signal to a desired amplitude for further signal processing. The image data signal is then processed by additional electrical components, such as an analog to digital converter.
While lock-in amplifiers are useful instruments for optical microscopy devices, such as the heterodyne detected optical image apparatus described above, this type of amplifier does exhibit some disadvantages. First, lock-in amplifiers exhibit a large thermal noise, which is detrimental to the signal to noise ratio (“SNR”) of the electrical output signal. As an example, the SNR at low laser power (as is used with live cell imaging) is limited by the electrical noise produced by the Johnson-Nyquist noise of the input impedance of the lock-in amplifier's input preamplifier. Attempting to improve the SNR by increasing the input impedance only worsens the SNR for a MHz-modulated signal due to the input capacitance of the lock-in amplifier, among other factors. Second, lock-in amplifiers typically process the input signal more slowly than is desired by most users. For example, the widely used SR844 digital lock-in amplifier offered by Stanford Research Systems has a minimum time constant of approximately 20 μs. At such a time constant, it takes tens of seconds to obtain an image of 512×512 pixels. Third, lock-in amplifiers are complex and expensive devices, which set a bottleneck for the wide use of heterodyne detected nonlinear optical microscopy.
Accordingly, further developments based on one or more of the above-described limitations are desirable for heterodyne detected optical imaging apparatus.