Infectious diseases are caused by microorganisms such as bacteria, viruses, fungi and parasites. Infectious diseases are major killer around the world especially in developing countries. Infectious diseases were responsible for 14.7 million deaths around the world in 2002 [¹] major portion of health care budget are allocated for diagnosis and treatment of infectious diseases. There are many diagnostic methods and techniques available for microorganisms associated diseases. These include morphological examination by microscopy, culture examination, biochemical tests, and histopathology approach. There are modern sophisticated methods are also available like PCR, ELISA, molecular DNA analysis. But, there are many limitations and drawbacks associated with these diagnostic techniques. These techniques are time consuming, laborious and require many reagents [²]. Also, some techniques lack high sensitivity and specificity which warrants the need for a new diagnostic technique with high sensitivity and specificity.

Current traditional diagnostic techniques and methods for diagnosis of microorganisms like bacteria take normally at least one day. Also, Antibiotic sensitivity testing is also required by physicians to choose specific antibiotic for treating infection. This sensitivity testing usually takes one more day. Bacteria are cultured for at least one day and then diagnosis is made. This causes delay in start of specific treatment. As a result physicians usually prescribe broad spectrum antibiotics which are unnecessary and very expensive for patients. Also, microorganisms have unique mechanisms to develop resistance for antimicrobial treatment. It justify for fast diagnosis of microorganisms and start of specific treatment as soon as possible.

Fluorescence spectroscopy seems to be promising diagnostic technique with fast and rapid diagnosis ability. Studies indicate high sensitivity and specificity rate which makes Fluorescence spectroscopy an ideal diagnostic tool for medical microbiology field. But, there is need for further studies and clinical trials to validate this new diagnostic technique.

At present, Fluorescence spectroscopy is being applied in medical microbiology field for various purposes. There are many studies which indicate that Fluorescence spectroscopy is promising diagnostic technique with high sensitivity and specificity for microorganisms associated diseases diagnosis with the help of spectroscopic fingerprints. Also, Fluorescence spectroscopy and Fluorescence correlation spectroscopy (FCS) may be applied to understand various pathophysiological steps of various microorganisms [³,⁴].

Fluorescence spectroscopy is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. The sample is excited by using a beam of light which results in emission of light of a lower energy resulting in an emission spectrum which is used to interpret results [⁵]. Fluorescence correlation spectroscopy (FCS), a technique basically used for spatial and temporal analysis of molecular interactions of extremely low concentrated biomolecules in solution. (Figure 1) FCS measures both the average number of molecules in the detection volume and the diffusion time of the molecules through the open detection volume [⁶]. As the diffusion speed is directly correlated with the molecular mass and shape of the fluorescent molecule, it is possible to study the complex formation between a small fluorescent labeled and a big unlabelled molecule [⁷].

Fluorescence correlation spectroscopy (FCS) use the basic principle that a fluorescing molecule shows a specific free diffusion velocity which is directly correlated with its size. So, bigger the molecule, slower it will diffuse through a given spherical volume. This basic phenomenon of molecules is used in FCS to study protein-protein interactions, attachment and many more. (Figure 1) Fluorescence Correlation Spectroscopy (FCS) uses statistical deviations of the fluctuations in fluorescence in order to study dynamic molecular events, such as diffusion or conformational fluctuations of bio molecules or artificial particles. (Figure 2) Mainly, the auto correlation function (ACF) is used to extract the number and diffusion coefficient of fluorescent particles diffusing through the focus volume. (Figure 3) These all properties of FCS make it an excellent diagnostic and research tool for many medically important diseases. Various properties of FCS make it an ideal tool for understanding various pathophysiological processes involved with microbial infectious diseases. An excellent advantage of FCS is that it requires very low concentrations and amounts of samples, as compared to routinely used techniques which require high concentration of diagnostic sample.

Tryptophan which is fluorophore in UV is present in both viruses and host bacterial protein. Indole group of tryptophan residues are major source of UV absorbance and emission in proteins. Tryptophan in pure water emits at 353 nm [⁸]. Tryptophan emission is strongly associated with its local environment. Many phenomena such as protein-protein association result in spectral shifts in tryptophan emission [⁸]. It is proposed that emission and excitation spectral differences may be due to presence of different environments of tryptophan residues in specific proteins of microorganism's cells [⁹].

At present, many studies reported successful application of Fluorescence spectroscopy as a diagnostic tool for different bacteria at genus, species and group level by use of spectral fingerprints [^10-12]. Spectral studies for black pigmented bacilli which are a group of oral bacteria showed significant difference in spectral signatures of each bacterium [¹²]. Fluorescent profiles of Bacteria which are responsible for otitis Media in children: S. pneumoniae, S. aureus, M. catarrhalis, and H. influenzae have been studied. These studies proved that each bacterium produce a different specific Fluorescence profile. The data indicate that it may be an excellent non invasive fluorescence based diagnostic technique for otitis media [¹²]. In another study; three different bacterial species (Escherichia coli, EC, Enterococcus faecalis, EF and Staphylococcus aureus, SA) were rapidly identified by autofluorescence spectrum differences coupled with Principal Components Analysis (PCA) technique. These studies proposed that bacteria can be rapidly diagnosed with sensitivity and specificity higher then 90% [¹³].

Fluorescence spectroscopy was utilized for pseudomonad taxonomic purpose at species and genus level [¹⁴]. Results proved that Fluorescence spectroscopy may be an excellent tool in polyphasic approach to pseudomonad taxonomy. This approach provide more information as compared to rRNA and DNA bacterial homology grouping as they provide more information about strain relatedness and good differentiation between strains which are difficult to differentiate on PCR and API 20NE identification methods [¹⁴].

Fungal infections are common in many diseases like diabetes, many types of cancers, endocrinopathies, and patients on prolonged antibiotics or immunosuppressive drugs. Diagnosis of fungal infection is made either by morphological examination of fungi or by biochemical and molecular biology techniques [¹⁵]. These techniques may not differentiate between different types of yeast. There are studies which have utilized spectroscopic fingerprints method for rapid diagnosis of different fungi such as yeast, Microsporum gypseum, Microsporum canis, Trichophyton schoenleinii, Trichophyton rubrum, Epidermophyton floccosum and Fusarium solani [⁹,¹⁶].

Studies indicate that Fluorescence spectroscopy may be a novel diagnostic tool to detect viruses. Also viral infections of cells can be monitored by Fluorescence spectroscopy [³]. These studies were carried out on viruses from cystovirus family and pseudomonad host cells. Tryptophan which is fluorophore in UV is present in both viruses and host bacterial protein. Within proteins, tryptophan structural environment is not same and this structural difference is responsible for specific spectroscopic signatures [³]. This property can be used to monitor viral attachment process and to study the release of progeny virus particles by analysis of tryptophan emission spectra during infection process.

In author's Lab, Fluorescence correlation spectroscopy (FCS) has been applied successfully to understand human rhino virus-receptor interaction [¹⁷]. These experiments provide informative data for understanding virus-receptor interactions. Fluorescence correlation spectroscopy (FCS) studies revealed different binding modes for an icosahedral virus along the five-fold symmetry axis. We proposed that Fluorescence correlation spectroscopy (FCS) may be a valuable technique to study various receptor binding affinities of viruses.