Scanning observation apparatus

1. A scanning observation apparatus comprising:

a scanning unit that spatially scans pulsed excitation light emitted from a light source at prescribed time intervals on a specimen;

a fluorescence detecting unit that detects fluorescence generated by exciting a fluorescent substance inside the specimen with the excitation light scanned by the scanning unit, in synchronization with the emission of the excitation light; and

a fluorescence correcting unit that subtracts, from a fluorescence intensity detected by the fluorescence detecting unit, an afterimage fluorescence component calculated on the basis of time-sequential fluorescence detected by the fluorescence detecting unit prior thereto, at each scanning position, to correct the fluorescence intensity at the scanning position,

wherein the fluorescence correcting unit corrects the fluorescence intensity on the basis of the following equations:

S O =L −1 ·S D

S

D

=

(

SD

1

SD

2

SD

3

⋮

SD

n

)

⁢

⁢

L

=

(

l

11

l

12

l

13

…

l

1

⁢

n

l

21

l

22

l

23

…

l

2

⁢

n

l

31

l

32

l

33

…

l

3

⁢

n

⋮

⋮

⋮

⋮

l

n

⁢

⁢

1

l

n

⁢

⁢

2

l

n

⁢

⁢

3

…

l

nn

)

⁢

⁢

S

O

=

(

SO

1

SO

2

SO

3

⋮

SO

n

)

{

MATH

⁢

⁢

1

}

 where

S O is a corrected fluorescence intensity,

SO m is an m-th corrected fluorescence intensity,

S D is a detected fluorescence intensity,

SD m is an m-th detected fluorescence intensity,

Δt is the time interval of the excitation light,

Tm is the fluorescence lifetime of a fluorescent dye, corresponding to the m-th detection,

L is a fluorescence lifetime matrix,

l ij =0 (i<j), and

lij=exp(((j−i)·Δt/T j ) (i≧j).

2. The scanning observation apparatus according to claim 1 , wherein the fluorescence correcting unit corrects the fluorescence intensity on the basis of the following equations:

SO m =SD m −SR m

SR m =exp(−Δ t/T )· SD m-1

where

SO m is the m-th corrected fluorescence intensity,

SD m is the m-th detected fluorescence intensity,

SR m is the m-th afterimage component,

Δt is the time interval of the excitation light, and

T is the fluorescence lifetime of the fluorescent dye.

3. The scanning observation apparatus according to claim 2 :

each pixel is formed of fluorescences generated by irradiation with pulsed excitation light a plurality of times;

each scanning line is formed of a plurality of the pixels;

one viewing field is formed of a plurality of divided viewing fields that are formed of a plurality of the scanning lines; and

in the case where, for each irradiation with pulsed excitation light one time, the divided viewing field serving as an irradiation target is switched in a prescribed order, the fluorescence correcting unit corrects the fluorescence intensity on the basis of the following equation:

SO

iq

=

∑

k

=

1

l

⁢

SO

ikq

⁢

⁢

⁢

{

=

∑

k

=

1

l

⁢

(

SD

ikq

-

exp

⁡

(

-

Δ

⁢

⁢

t

/

T

)

·

SD

ik

⁡

(

q

-

1

)

)

(

2

≤

q

≤

r

)

=

∑

k

=

1

l

⁢

SD

ik

⁢

⁢

1

-

exp

⁡

(

-

Δ

⁢

⁢

t

/

T

)

·

(

SD

(

i

-

1

)

⁢

lr

+

∑

k

=

1

l

-

1

⁢

SD

ikr

)

(

q

=

1

)

where

l is the number of detections of the fluorescences forming each pixel,

r is the total number of divided viewing fields,

SO iq is the corrected fluorescence intensity in the i-th pixel in the q-th divided viewing field,

SO ikq is the corrected fluorescence intensity, due to the k-th pulsed excitation light irradiation, in the i-th pixel in the q-th divided viewing field,

SD ikq is the detected fluorescence intensity, due to the k-th pulsed excitation light irradiation, in the i-th pixel in the q-th divided viewing field,

SD ikq is the detected fluorescence intensity, due to the k-th pulsed excitation light irradiation, in the i-th pixel in the first (initial) divided viewing field, and

SD (i-1)|r is the detected fluorescence intensity, due to the l-th (final) pulsed excitation light irradiation, in the (i−1)-th pixel in the r-th (final) divided viewing field.

4. The scanning observation apparatus according to claim 2 , wherein:

each pixel is formed of fluorescences generated by irradiation with pulsed excitation light a plurality of times;

each scanning line is formed of a plurality of the pixels;

one viewing field is formed of a plurality of divided viewing fields formed of a plurality of the scanning lines; and

in the case where, for each irradiation with pulsed excitation light one time, the divided viewing field serving as an irradiation target is switched in a prescribed order, the fluorescence correcting unit corrects the fluorescence intensity on the basis of the following equation:

SO

iq

⁢

{

=

∑

k

=

1

l

⁢

(

SD

ikq

-

exp

⁡

(

-

Δ

⁢

⁢

t

/

T

)

·

∑

k

=

1

l

⁢

SD

ik

⁡

(

q

-

1

)

)

=

SD

iq

-

exp

⁡

(

-

Δ

⁢

⁢

t

/

T

)

·

SD

i

⁡

(

q

-

1

)

(

2

≤

q

≤

r

)

(

q

=

1

)

where

l is the number of detections of the fluorescences forming each pixel,

r is the total number of divided viewing fields,

SO iq is the corrected fluorescence intensity in the i-th pixel in the q-th divided viewing field,

SO ikq is the corrected fluorescence intensity, due to the k-th pulsed excitation light irradiation, in the i-th pixel in the q-th divided viewing field,

SD iq is the detected fluorescence intensity in the i-th pixel in the q-th divided viewing field,

SD il is the detected fluorescence intensity in the i-th pixel in the first (initial) divided viewing field, and

SD ir is the detected fluorescence intensity in the i-th pixel in the r-th (final) divided viewing field.